Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

ABSTRACT

A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

This application is a continuation of 08/621,859 filed Mar. 25, 1996,the disclosure of which is incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to a method for the production ofpolynucleotides conferring a desired phenotype and/or encoding a proteinhaving an advantageous predetermined property which is selectable or canbe screened for. In an aspect, the method is used for generating andselecting or screening for desired nucleic acid fragments encodingmutant proteins.

BACKGROUND AND DESCRIPTION OF RELATED ART

The complexity of an active sequence of a biological macromolecule, e.g.proteins, DNA etc., has been called its information content ("IC"; 5-9).The information content of a protein has been defined as the resistanceof the active protein to amino acid sequence variation, calculated fromthe minimum number of invariable amino acids (bits) required to describea family of related sequences with the same function (9, 10). Proteinsthat are sensitive to random mutagenesis have a high informationcontent. In 1974, when this definition was coined, protein diversityexisted only as taxonomic diversity.

Molecular biology developments such as molecular libraries have allowedthe identification of a much larger number of variable bases, and evento select functional sequences from random libraries. Most residues canbe varied, although typically not all at the same time, depending oncompensating changes in the context. Thus a 100 amino acid protein cancontain only 2,000 different mutations, but 20¹⁰⁰ possible combinationsof mutations.

Information density is the Information Content/unit length of asequence. Active sites of enzymes tend to have a high informationdensity. By contrast, flexible linkers in enzymes have a low informationdensity (8).

Current methods in widespread use for creating mutant proteins in alibrary format are error-prone polymerase chain reaction (11, 12, 19)and cassette mutagenesis (8, 20, 21, 22, 40, 41, 42), in which thespecific region to be optimized is replaced with a syntheticallymutagenized oligonucleotide. Alternatively, mutator strains of hostcells have been employed to add mutational frequency (Greener andCallahan (1995) Strategies in Mol. Biol. 7: 32). In each case, a `mutantcloud` (4) is generated around certain sites in the original sequence.

Error-prone PCR uses low-fidelity polymerization conditions to introducea low level of point mutations randomly over a long sequence. Errorprone PCR can be used to mutagenize a mixture of fragments of unknownsequence. However, computer simulations have suggested that pointmutagenesis alone may often be too gradual to allow the block changesthat are required for continued sequence evolution. The publishederror-prone PCR protocols are generally unsuited for reliableamplification of DNA fragments greater than 0.5 to 1.0 kb, limitingtheir practical application. Further, repeated cycles of error-prone PCRlead to an accumulation of neutral mutations, which, for example, maymake a protein immunogenic.

In oligonucleotide-directed mutagenesis, a short sequence is replacedwith a synthetically mutagenized oligonucleotide. This approach does notgenerate combinations of distant mutations and is thus not significantlycombinatorial. The limited library size relative to the vast sequencelength means that many rounds of selection are unavoidable for proteinoptimization. Mutagenesis with synthetic oligonucleotides requiressequencing of individual clones after each selection round followed bygrouping into families, arbitrarily choosing a single family, andreducing it to a consensus motif, which is resynthesized and reinsertedinto a single gene followed by additional selection. This processconstitutes a statistical bottleneck, it is labor intensive and notpractical for many rounds of mutagenesis.

Error-prone PCR and oligonucleotide-directed mutagenesis are thus usefulfor single cycles of sequence fine tuning but rapidly become limitingwhen applied for multiple cycles.

Error-prone PCR can be used to mutagenize a mixture of fragments ofunknown sequence (11, 12). However, the published error-prone PCRprotocols (11, 12) suffer from a low processivity of the polymerase.Therefore, the protocol is very difficult to employ for the randommutagenesis of an average-sized gene. This inability limits thepractical application of error-prone PCR.

Another serious limitation of error-prone PCR is that the rate ofdown-mutations grows with the information content of the sequence. At acertain information content, library size, and mutagenesis rate, thebalance of down-mutations to up-mutations will statistically prevent theselection of further improvements (statistical ceiling).

Finally, repeated cycles of error-prone PCR will also lead to theaccumulation of neutral mutations, which can affect, for example,immunogenicity but not binding affinity.

Thus error-prone PCR was found to be too gradual to allow the blockchanges that are required for continued sequence evolution (1, 2).

In cassette mutagenesis, a sequence block of a single template istypically replaced by a (partially) randomized sequence. Therefore, themaximum information content that can be obtained is statisticallylimited by the number of random sequences (i.e., library size). Thisconstitutes a statistical bottleneck, eliminating other sequencefamilies which are not currently best, but which may have greater longterm potential.

Further, mutagenesis with synthetic oligonucleotides requires sequencingof individual clones after each selection round (20). Therefore, thisapproach is tedious and is not practical for many rounds of mutagenesis.

Error-prone PCR and cassette mutagenesis are thus best suited and havebeen widely used for fine-tuning areas of comparatively low informationcontent. An example is the selection of an RNA ligase ribozyme from arandom library using many rounds of amplification by error-prone PCR andselection (13).

It is becoming increasingly clear our scientific tools for the design ofrecombinant linear biological sequences such as protein, RNA and DNA arenot suitable for generating the necessary sequence diversity needed tooptimize many desired properties of a macromolecule or organism. Findingbetter and better mutants depends on searching more and more sequenceswithin larger and larger libraries, and increasing numbers of cycles ofmutagenic amplification and selection are necessary. However asdiscussed above, the existing mutagenesis methods that are in widespreaduse have distinct limitations when used for repeated cycles.

Evolution of most organisms occurs by natural selection and sexualreproduction. Sexual reproduction ensures mixing and combining of thegenes of the offspring of the selected individuals. During meiosis,homologous chromosomes from the parents line up with one another andcross-over part way along their length, thus swapping genetic material.Such swapping or shuffling of the DNA allows organisms to evolve morerapidly (1, 2). In sexual recombination, because the inserted sequenceswere of proven utility in a homologous environment, the insertedsequences are likely to still have substantial information content oncethey are inserted into the new sequence.

Marton et al.,(27) describes the use of PCR in vitro to monitorrecombination in a plasmid having directly repeated sequences. Marton etal. discloses that recombination will occur during PCR as a result ofbreaking or nicking of the DNA. This will give rise to recombinantmolecules. Meyerhans et al. (23) also disclose the existence of DNArecombination during in vitro PCR.

The term Applied Molecular Evolution ("AME") means the application of anevolutionary design algorithm to a specific, useful goal. While manydifferent library formats for AME have been reported for polynucleotides(3, 11-14), peptides and proteins (phage (15-17), laci (18) andpolysomes, in none of these formats has recombination by randomcross-overs been used to deliberately create a combinatorial library.

Theoretically there are 2,000 different single mutants of a 100 aminoacid protein. A protein of 100 amino acids has 20¹⁰⁰ possiblecombinations of mutations, a number which is too large to exhaustivelyexplore by conventional methods. It would be advantageous to develop asystem which would allow the generation and screening of all of thesepossible combination mutations.

Winter and coworkers (43,44) have utilized an in vivo site specificrecombination system to combine light chain antibody genes with heavychain antibody genes for expression in a phage system. However, theirsystem relies on specific sites of recombination and thus is limited.Hayashi et al. (48) report simultaneous mutagenesis of antibody CDRregions in single chain antibodies (scFv) by overlap extension and PCR.

Caren et al. (45) describe a method for generating a large population ofmultiple mutants using random in vivo recombination. However, theirmethod requires the recombination of two different libraries ofplasmids, each library having a different selectable marker. Thus themethod is limited to a finite number of recombinations equal to thenumber of selectable markers existing, and produces a concomitant linearincrease in the number of marker genes linked to the selectedsequence(s). Caren et al. does not describe the use of multipleselection cycles; recombination is used solely to construct largerlibraries.

Calogero et al. (46) and Galizzi et al. (47) report that in vivorecombination between two homologous but truncated insect-toxin genes ona plasmid can produce a hybrid gene. Radman et al. (49) report in vivorecombination of substantially mismatched DNA sequences in a host cellhaving defective mismatch repair enzymes, resulting in hybrid moleculeformation.

It would be advantageous to develop a method for the production ofmutant proteins which method allowed for the development of largelibraries of mutant nucleic acid sequences which were easily searched.The invention described herein is directed to the use of repeated cyclesof point mutagenesis, nucleic acid shuffling and selection which allowfor the directed molecular evolution in vitro of highly complex linearsequences, such as proteins through random recombination.

Accordingly, it would be advantageous to develop a method which allowsfor the production of large libraries of mutant DNA, RNA or proteins andthe selection of particular mutants for a desired goal. The inventiondescribed herein is directed to the use of repeated cycles ofmutagenesis, in vivo recombination and selection which allow for thedirected molecular evolution in vivo and in vitro of highly complexlinear sequences, such as DNA, RNA or proteins through recombination.

Further advantages of the present invention will become apparent fromthe following description of the invention with reference to theattached drawings.

SUMMARY OF THE INVENTION

The present invention is directed to a method for generating a selectedpolynucleotide sequence or population of selected polynucleotidesequences, typically in the form of amplified and/or clonedpolynucleotides, whereby the selected polynucleotide sequence(s) possessa desired phenotypic characteristic (e.g., encode a polypeptide, promotetranscription of linked polynucleotides, bind a protein, and the like)which can be selected for. One method of identifying polypeptides thatpossess a desired structure or functional property, such as binding to apredetermined biological macromolecule (e.g., a receptor), involves thescreening of a large library of polypeptides for individual librarymembers which possess the desired structure or functional propertyconferred by the amino acid sequence of the polypeptide.

In a general aspect, the invention provides a method, termed "sequenceshuffling", for generating libraries of recombinant polynucleotideshaving a desired characteristic which can be selected or screened for.Libraries of recombinant polynucleotides are generated from a populationof related-sequence polynucleotides which comprise sequence regionswhich have substantial sequence identity and can be homologouslyrecombined in vitro or in vivo. In the method, at least two species ofthe related-sequence polynucleotides are combined in a recombinationsystem suitable for generating sequence-recombined polynucleotides,wherein said sequence-recombined polynucleotides comprise a portion ofat least one first species of a related-sequence polynucleotide with atleast one adjacent portion of at least one second species of arelated-sequence polynucleotide. Recombination systems suitable forgenerating sequence-recombined polynucleotides can be either: (1) invitro systems for homologous recombination or sequence shuffling viaamplification or other formats described herein, or (2) in vivo systemsfor homologous recombination or site-specific recombination as describedherein. The population of sequence-recombined polynucleotides comprisesa subpopulation of polynucleotides which possess desired or advantageouscharacteristics and which can be selected by a suitable selection orscreening method. The selected sequence-recombined polynucleotides,which are typically related-sequence polynucleotides, can then besubjected to at least one recursive cycle wherein at least one selectedsequence-recombined polynucleotide is combined with at least onedistinct species of related-sequence polynucleotide (which may itself bea selected sequence-recombined polynucleotide) in a recombination systemsuitable for generating sequence-recombined polynucleotides, such thatadditional generations of sequence-recombined polynucleotide sequencesare generated from the selected sequence-recombined polynucleotidesobtained by the selection or screening method employed. In this manner,recursive sequence recombination generates library members which aresequence-recombined polynucleotides possessing desired characteristics.Such characteristics can be any property or attribute capable of beingselected for or detected in a screening system, and may includeproperties of: an encoded protein, a transcriptional element, a sequencecontrolling transcription, RNA processing, RNA stability, chromatinconformation, translation, or other expression property of a gene ortransgene, a replicative element, a protein-binding element, or thelike, such as any feature which confers a selectable or detectableproperty.

The present invention provides a method for generating libraries ofdisplayed polypeptides or displayed antibodies suitable for affinityinteraction screening or phenotypic screening. The method comprises (1)obtaining a first plurality of selected library members comprising adisplayed polypeptide or displayed antibody and an associatedpolynucleotide encoding said displayed polypeptide or displayedantibody, and obtaining said associated polynucleotides or copiesthereof wherein said associated polynucleotides comprise a region ofsubstantially identical sequence, optionally introducing mutations intosaid polynucleotides or copies, and (2) pooling and fragmenting, bynuclease digestion, partial extension PCR amplification, PCR stuttering,or other suitable fragmenting means, typically producing randomfragments or fragment equivalents, said associated polynucleotides orcopies to form fragments thereof under conditions suitable for PCRamplification, performing PCR amplification and optionally mutagenesis,and thereby homologously recombining said fragments to form a shuffledpool of recombined polynucleotides, whereby a substantial fraction(e.g., greater than 10 percent) of the recombined polynucleotides ofsaid shuffled pool are not present in the first plurality of selectedlibrary members, said shuffled pool composing a library of displayedpolypeptides or displayed antibodies suitable for affinity interactionscreening. Optionally, the method comprises the additional step ofscreening the library members of the shuffled pool to identifyindividual shuffled library members having the ability to bind orotherwise interact (e.g., such as catalytic antibodies) with apredetermined macromolecule, such as for example a proteinaceousreceptor, peptide, oligosaccharide, virion, or other predeterminedcompound or structure. The displayed polypeptides, antibodies,peptidomimetic antibodies, and variable region sequences that areidentified from such libraries can be used for therapeutic, diagnostic,research, and related purposes (e.g., catalysts, solutes for increasingosmolarity of an aqueous solution, and the like), and/or can besubjected to one or more additional cycles of shuffling and/or affinityselection. The method can be modified such that the step of selecting isfor a phenotypic characteristic other than binding affinity for apredetermined molecule (e.g., for catalytic activity, stability,oxidation resistance, drug resistance, or detectable phenotype conferredon a host cell).

In one embodiment, the first plurality of selected library members isfragmented and homologously recombined by PCR in vitro. Fragmentgeneration is by nuclease digestion, partial extension PCRamplification, PCR stuttering, or other suitable fragmenting means, suchas described herein. Stuttering is fragmentation by incompletepolymerase extension of templates. A recombination format based on veryshort PCR extension times was employed to create partial PCR products,which continue to extend off a different template in the next (andsubsequent) cycle(s).

In one embodiment, the first plurality of selected library members isfragmented in vitro, the resultant fragments transferred into a hostcell or organism and homologously recombined to form shuffled librarymembers in vivo.

In one embodiment, the first plurality of selected library members iscloned or amplified on episomally replicable vectors, a multiplicity ofsaid vectors is transferred into a cell and homologously recombined toform shuffled library members in vivo.

In one embodiment, the first plurality of selected library members isnot fragmented, but is cloned or amplified on an episomally replicablevector as a direct repeat or indirect (or inverted) repeat, which eachrepeat comprising a distinct species of selected library membersequence, said vector is transferred into a cell and homologouslyrecombined by intra-vector or inter-vector recombination to formshuffled library members in vivo.

In an embodiment, combinations of in vitro and in vivo shuffling areprovided to enhance combinatorial diversity.

The present invention provides a method for generating libraries ofdisplayed antibodies suitable for affinity interaction screening. Themethod comprises (1) obtaining a first plurality of selected librarymembers comprising a displayed antibody and an associated polynucleotideencoding said displayed antibody, and obtaining said associatedpolynucleotides or copies thereof, wherein said associatedpolynucleotides comprise a region of substantially identical variableregion framework sequence, and (2) pooling and fragmenting saidassociated polynucleotides or copies to form fragments thereof underconditions suitable for PCR amplification and thereby homologouslyrecombining said fragments to form a shuffled pool of recombinedpolynucleotides comprising novel combinations of CDRs, whereby asubstantial fraction (e.g., greater than 10 percent) of the recombinedpolynucleotides of said shuffled pool comprise CDR combinations whichare not present in the first plurality of selected library members, saidshuffled pool composing a library of displayed antibodies comprising CDRpermutations and suitable for affinity interaction screening.Optionally, the shuffled pool is subjected to affinity screening toselect shuffled library members which bind to a predetermined epitope(antigen) and thereby selecting a plurality of selected shuffled librarymembers. Optionally, the plurality of selected shuffled library memberscan be shuffled and screened iteratively, from 1 to about 1000 cycles oras desired until library members having a desired binding affinity areobtained.

Accordingly, one aspect of the present invention provides a method forintroducing one or more mutations into a template double-strandedpolynucleotide, wherein the template double-stranded polynucleotide hasbeen cleaved or PCR amplified (via partial extension or stuttering) intorandom fragments of a desired size, by adding to the resultantpopulation of double-stranded fragments one or more single ordouble-stranded oligonucleotides, wherein said oligonucleotides comprisean area of identity and an area of heterology to the templatepolynucleotide; denaturing the resultant mixture of double-strandedrandom fragments and oligonucleotides into single-stranded fragments;incubating the resultant population of single-stranded fragments with apolymerase under conditions which result in the annealing of saidsingle-stranded fragments at regions of identity between thesingle-stranded fragments and formation of a mutagenized double-strandedpolynucleotide; and repeating the above steps as desired.

In another aspect the present invention is directed to a method ofproducing recombinant proteins having biological activity by treating asample comprising double-stranded template polynucleotides encoding awild-type protein under conditions which provide for the cleavage ofsaid template polynucleotides into random double-stranded fragmentshaving a desired size; adding to the resultant population of randomfragments one or more single or double-stranded oligonucleotides,wherein said oligonucleotides comprise areas of identity and areas ofheterology to the template polynucleotide; denaturing the resultantmixture of double-stranded fragments and oligonucleotides intosingle-stranded fragments; incubating the resultant population ofsingle-stranded fragments with a polymerase under conditions whichresult in the annealing of said single-stranded fragments at the areasof identity and formation of a mutagenized double-strandedpolynucleotide; repeating the above steps as desired; and thenexpressing the recombinant protein from the mutagenized double-strandedpolynucleotide.

A third aspect of the present invention is directed to a method forobtaining a chimeric polynucleotide by treating a sample comprisingdifferent double-stranded template polynucleotides wherein saiddifferent template polynucleotides contain areas of identity and areasof heterology under conditions which provide for the cleavage of saidtemplate polynucleotides into random double-stranded fragments of adesired size; denaturing the resultant random double-stranded fragmentscontained in the treated sample into single-stranded fragments;incubating the resultant single-stranded fragments with polymerase underconditions which provide for the annealing of the single-strandedfragments at the areas of identity and the formation of a chimericdouble-stranded polynucleotide sequence comprising templatepolynucleotide sequences; and repeating the above steps as desired.

A fourth aspect of the present invention is directed to a method ofreplicating a template polynucleotide by combining in vitrosingle-stranded template polynucleotides with small randomsingle-stranded fragments resulting from the cleavage and denaturationof the template polynucleotide, and incubating said mixture of nucleicacid fragments in the presence of a nucleic acid polymerase underconditions wherein a population of double-stranded templatepolynucleotides is formed.

The invention also provides the use of polynucleotide shuffling, invitro and/or in vivo to shuffle polynucleotides encoding polypeptidesand/or polynucleotides comprising transcriptional regulatory sequences.

The invention also provides the use of polynucleotide shuffling toshuffle a population of viral genes (e.g., capsid proteins, spikeglycoproteins, polymerases, proteases, etc.) or viral genomes (e.g.,paramyxoviridae, orthomyxoviridae, herpesviruses, retroviruses,reoviruses, rhinoviruses, etc.). In an embodiment, the inventionprovides a method for shuffling sequences encoding all or portions ofimmunogenic viral proteins to generate novel combinations of epitopes aswell as novel epitopes created by recombination; such shuffled viralproteins may comprise epitopes or combinations of epitopes which arelikely to arise in the natural environment as a consequence of viralevolution (e.g., such as recombination of influenza virus strains).

The invention also provides the use of polynucleotide shuffling toshuffle a population of protein variants, such as taxonomically-related,structurally-related, and/or functionally-related enzymes and/or mutatedvariants thereof to create and identify advantageous novel polypeptides,such as enzymes having altered properties of catalysis, temperatureprofile, stability, oxidation resistance, or other desired feature whichcan be selected for. Methods suitable for molecular evolution anddirected molecular evolution are provided. Methods to focus selectionpressure(s) upon specific portions of polynucleotides (such as a segmentof a coding region) are provided.

The invention also provides a method suitable for shufflingpolynucleotide sequences for generating gene therapy vectors andreplication-defective gene therapy constructs, such as may be used forhuman gene therapy, including but not limited to vaccination vectors forDNA-based vaccination, as well as anti-neoplastic gene therapy and othergene therapy formats.

The invention provides a method for generating an enhanced greenfluorescent protein (GFP) and polynucleotides encoding same, comprisingperforming DNA shuffling on a GFP encoding expression vector andselecting or screening for variants having an enhanced desired property,such as enhanced fluorescence. In a variation, an embodiment comprises astep of error-prone or mutagenic amplification, propagation in a mutatorstrain (e.g., a host cell having a hypermutational phenotype; mut^(L),etc.; yeast strains such as those described in Klein (1995) Progr. Nucl.Acid Res. Mol. Biol. 51: 217, incorporated herein by reference),chemical mutagenesis, or site-directed mutagenesis. In an embodiment,the enhanced GFP protein comprises a point mutation outside thechromophore region (amino acids 64-69), preferably in the region fromamino acid 100 to amino acid 173, with specific preferred embodiments atresidue 100, 154, and 164; typically, the mutation is a substitutionmutation, such as F100S, M154T or V164A. In an embodiment, the mutationsubstitutes a hydrophilic residue for a hydrophobic residue. In anembodiment, multiple mutations are present in the enhanced GFP proteinand its encoding polynucleotide. The invention also provides the use ofsuch an enhanced GFP protein, such as for a diagnostic reporter forassays and high throughput screening assays and the like.

The invention also provides for improved embodiments for performing invitro sequence shuffling. In one aspect, the improved shuffling methodincludes the addition of at least one additive which enhances the rateor extent of reannealing or recombination of related-sequencepolynucleotides. In an embodiment, the additive is polyethylene glycol(PEG), typically added to a shuffling reaction to a final concentrationof 0.1 to 25 percent, often to a final concentration of 2.5 to 15percent, to a final concentration of about 10 percent. In an embodiment,the additive is dextran sulfate, typically added to a shuffling reactionto a final concentration of 0.1 to 25 percent, often at about 10percent. In an embodiment, the additive is an agent which reducessequence specificity of reannealing and promotes promiscuoushybridization and/or recombination in vitro. In an alternativeembodiment, the additive is an agent which increases sequencespecificity of reannealing and promotes high fidelity hybridizationand/or recombination in vitro. Other long-chain polymers which do notinterfere with the reaction may also be used (e.g.,polyvinylpyrrolidone, etc.).

In one aspect, the improved shuffling method includes the addition of atleast one additive which is a cationic detergent. Examples of suitablecationic detergents include but are not limited to:cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide(DTAB), and tetramethylammonium chloride (TMAC), and the like.

In one aspect, the improved shuffling method includes the addition of atleast one additive which is a recombinogenic protein that catalyzes ornon-catalytically enhances homologous pairing and/or strand exchange invitro. Examples of suitable recombinogenic proteins include but are notlimited to: E. coli recA protein, the T4 uvsX protein, the redl proteinfrom Ustilago maydis, other recA family recombinases from other species,single strand binding protein (SSB), ribonucleoprotein A1, and the like.Shuffling can be used to improve one or more properties of arecombinogenic protein; for example, mutant sequences encoding recA canbe shuffled and improved heat-stable variants selected by recursivesequence recombination.

Non-specific (general recombination) recombinases such as TopoisomeraseI, Topoisomerase II (Tse et al. (1980) J. Biol. Chem. 255: 5560; Trasket al. (1984) EMBO J. 3: 671, incorporated herein by reference) and thelike can be used to catalyze in vitro recombination reactions to shufflea plurality of related sequence polynucelotide species by the recursivemethods of the invention.

In one aspect, the improved shuffling method includes the addition of atleast one additive which is an enzyme having an exonuclease activitywhich is active at removing non-templated nucleotides introduced at 3'ends of product polynucleotides in shuffling amplification reactionscatalyzed by a non-proofreading polymerase. An example of a suitableenzyme having an exonuclease activity includes but is not limited to Pfupolymerase. Other suitable polymerases include, but are not limited to:Thermus flavus DNA polymerase (Tfl)

Thermus thermophilus DNA polymerase (Tth)

Thermococcus litoralis DNA polymerase (Tli, Vent)

Pyrococcus Woesei DNA polymerase (Pwo)

Thermotoga maritima DNA polymerase (UltMa)

Thermus brockianus DNA polymerase (Thermozyme)

Pyrococcus furiosus DNA polymerase (Pfu)

Thermococcus sp. DNA polymerase (9° Nm)

Pyrococcus sp. DNA polymerase (`Deep Vent`)

Bacteriophage T4 DNA polymerase

Bacteriophage T7 DNA polymerase

E. coli DNA polymerase I (native and Klenow)

E. coli DNA polymerase III.

In an aspect, the improved shuffling method comprises the modificationwherein at least one cycle of amplification (i.e., extension with apolymerase) of reannealed fragmented library member polynucleotides isconducted under conditions which produce a substantial fraction,typically at least 20 percent or more, of incompletely extendedamplification products. The amplification products, including theincompletely extended amplification products are denatured and subjectedto at least one additional cycle of reannealing and amplification. Thisvariation, wherein at least one cycle of reannealing and amplificationprovides a substantial fraction of incompletely extended products, istermed "stuttering" and in the subsequent amplification round theincompletely extended products reanneal to and prime extension ondifferent sequence-related template species.

In an aspect, the improved shuffling method comprises the modificationwherein at least one cycle of amplification is conducted using acollection of overlapping single-stranded DNA fragments of varyinglengths corresponding to a first polynucleotide species or set ofrelated-sequence polynucleotide species, wherein each overlappingfragment can each hybridize to and prime polynucleotide chain extensionfrom a second polynucleotide species serving as a template, thus formingsequence-recombined polynucleotides, wherein said sequence-recombinedpolynucleotides comprise a portion of at least one first polynucleotidespecies with an adjacent portion of the second polynucleotide specieswhich serves as a template. In a variation, the second polynucleotidespecies serving as a template contains uracil (i.e., a Kunkel-typetemplate) and is substantially non-replicable in cells. This aspect ofthe invention can also comprise at least two recursive cycles of thisvariation.

In an embodiment, PCR can be conducted wherein the nucleotide mixcomprises a nucleotide species having uracil as the base. The PCRproduct(s) can then be fragmented by digestion with UDG-glycosylasewhich produces strand breaks. The fragment size can be controlled by thefraction of uracil-containing NTP in the PCR mix.

In an aspect, the improved shuffling method comprises the modificationwherein at least one cycle of amplification is conducted with anadditive or polymerase in suitable conditions which promote templateswitching. In an embodiment where Taq polymerase is employed foramplification, addition of recA or other polymerases (e.g., viralpolymerases, reverse transcriptase) enhances template switching.Template-switching can also be increased by increasing the DNA templateconcentration, among other means known by those skilled in the art.

In an embodiment of the general method, libraries of sequence-recombinedpolynucleotides are generated from sequence-related polynucleotideswhich are naturally-occurring genes or alleles of a gene. In thisaspect, at least two naturally-occurring genes and/or alleles whichcomprise regions of at least 50 consecutive nucleotides which have atleast 70 percent sequence identity, preferably at least 90 percentsequence identity, are selected from a pool of gene sequences, such asby hybrid selection or via computerized sequence analysis using sequencedata from a database. In an aspect, at least three naturally-occurringgenes and/or alleles which comprise regions of at least 50 consecutivenucleotides which have at least 70 percent sequence identity,prefereably at least 90 percent sequence identity, are selected from apool of gene sequences, such as by hybrid selection or via computerizedsequence analysis using sequence data from a database. The selectedsequences are obtained as polynucleotides, either by cloning or via DNAsynthesis, and shuffled by any of the various embodiments of theinvention.

In an embodiment of the invention, multi-pool shuffling is performed.Shuffling of multiple pools of polynucleotide sequences allows eachseparate pool to generate a different combinatorial solution to producethe desired property. In this variation, the pool of parentalpolynucleotides sequences (or any subsequent shuffled library orselected pool of library members) is subdivided (or segregated) into twoor more discrete pools of sequences and are separately subjected to oneor more rounds of recursive sequence recombination and selection (orscreening). If desired, optionally, selected library members from eachseparate pool may be recombined (integrated) in latter rounds ofshuffling. Alternatively, multiple separate parental pools may be used.Inbreeding, wherein selected (or screened) library members within a poolare crossed with each other by the recursive sequence recombinationmethods of the invention, can be performed, alone or in combination withoutbreeding, wherein library members of different pools are crossed witheach other by the recursive sequence recombination methods of theinvention.

In an embodiment of the invention, the method comprises the further stepof removing non-shuffled products (e.g., parental sequences) fromsequence-recombined polynucleotides produced by any of the disclosedshuffling methods. Non-shuffled products can be removed or avoided byperforming amplification with: (1) a first PCR primer which hybridizesto a first parental polynucleotide species but does not substantiallyhybridize to a second parental polynucleotide species, and (2) a secondPCR primer which hybridizes to a second parental polynucleotide speciesbut does not substantially hybridize to the first parentalpolynucleotide species, such that amplification occurs from templatescomprising the portion of the first parental sequence which hybridizesto the first PCR primer and also comprising the portion of the secondparental sequence which hybridizes to the second PCR primer, thus onlysequence-recombined polynucleotides are amplified.

The invention also provides for alternative embodiments for performingin vivo sequence shuffling. In one aspect, the alternative shufflingmethod includes the use of inter-plasmidic recombination, whereinlibraries of sequence-recombined polynucleotide sequences are obtainedby genetic recombination in vivo of compatible or non-compatiblemulticopy plasmids inside suitable host cells. When non-compatibleplasmids are used, typically each plasmid type has a distinct selectablemarker and selection for retention of each desired plasmid type isapplied. The related-sequence polynucleotide sequences to be recombinedare separately incorporated into separately replicable multicopyvectors, typically bacterial plasmids each having a distinct andseparately selectable marker gene (e.g., a drug resistance gene).Suitable host cells are transformed with both species of plasmid andcells expressing both selectable marker genes are selected andsequence-recombined sequences are recovered and can be subjected toadditional rounds of shuffling by any of the means described herein.

In one aspect, the alternative shuffling method includes the use ofintra-plasmidic recombination, wherein libraries of sequence-recombinedpolynucleotide sequences are obtained by genetic recombination in vivoof direct or inverted sequence repeats located on the same plasmid. In avariation, the sequences to be recombined are flanked by site-specificrecombination sequences and the polynucleotides are present in asite-specific recombination system, such as an integron (Hall andCollins (1995) Mol. Microbiol. 15: 593, incorporated herein byreference) and can include insertion sequences, transposons (e.g., IS1),and the like. Introns have a low rate of natural mobility and can beused as mobile genetic elements both in prokaryotes and eukaryotes.Shuffling can be used to improve the performance of mobile geneticelements. These high frequency recombination vehicles can be used forthe rapid optimization of large sequences via transfer of large sequenceblocks. Recombination between repeated, interspersed, and diverged DNAsequences, also called "homeologous" sequences, is typically suppressedin normal cells. However, in MutL and MutS cells, this suppression isrelieved and the rate of intrachromosomal recombination is increased(Petit et al. (1996) Genetics 129: 327, incorporated herein byreference).

In an aspect of the invention, mutator strains of host cells are used toenhance recombination of more highly mismatched sequence-relatedpolynucleotides. Bacterials strains such as MutL, MutS, MutT, or MutH orother cells expressing the Mut proteins (XL-1red; Stratagene, San Diego,Calif.) can be used as host cells for shuffling of sequence-relatedpolynucleotides by in vivo recombination. Other mutation-prone host celltypes can also be used, such as those having a proofreading-defectivepolymerase (Foster et al. (1995) Proc. Natl. Acad. Sci. (U.S.A.) 92:7951, incorporated herein by reference). Mutator strains of yeast can beused, as can hypermutational mammalian cells, including ataxiatelangiectasia cells, such as described in Luo et al. (1996) J. Biol.Chem. 271: 4497, incorporated herein by reference.

Other in vivo and in vitro mutagenic formats can be employed, includingadministering chemical or radiological mutagens to host cells. Examplesof such mutagens include but are not limited to: MNU, ENU, MNNG,nitrosourea, BuDR, and the like. Ultraviolet light can also be used togenerate mutations and/or to enhance the rate of recombination, such asby irradiation of cells used to enhance in vivo recombination. Ionizingradiation and clastogenic agents can also be used to enhance mutationalfrequency and/or to enhance recombination and/or to effectpolynucleotide fragmentation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram comparing mutagenic shuffling overerror-prone PCR; (a) the initial library; (b) pool of selected sequencesin first round of affinity selection; (d) in vitro recombination of theselected sequences (`shuffling`); (f) pool of selected sequences insecond round of affinity selection after shuffling; (c) error-prone PCR;(e) pool of selected sequences in second round of affinity selectionafter error-prone PCR.

FIG. 2 illustrates the reassembly of a 1.0 kb LacZ alpha gene fragmentfrom 10-50 bp random fragments. (a) Photograph of a gel of PCR amplifiedDNA fragment having the LacZ alpha gene. (b) Photograph of a gel of DNAfragments after digestion with DNAseI. (c) Photograph of a gel of DNAfragments of 10-50 bp purified from the digested LacZ alpha gene DNAfragment; (d) Photograph of a gel of the 10-50 bp DNA fragments afterthe indicated number of cycles of DNA reassembly; (e) Photograph of agel of the recombination mixture after amplification by PCR withprimers.

FIGS. 3A and 3B show a a schematic illustration of the LacZ alpha genestop codon mutants and their DNA sequences. The boxed regions areheterologous areas, serving as markers. The stop codons are located insmaller boxes or underlined. "+" indicates a wild-type gene and "-"indicates a mutated area in the gene.

FIG. 4 is a schematic illustration of the introduction or spiking of asynthetic oligonucleotide into the reassembly process of the LacZ alphagene.

FIGS. 5A and 5B illustrate the regions of homology between a murineIL1-B gene (M) and a human IL1-B gene (H) with E. coli codon usage.Regions of heterology are boxed. The "₋₋ |⁻⁻ " indicate crossoversobtained upon the shuffling of the two genes.

FIG. 6 is a schematic diagram of the antibody CDR shuffling model systemusing the scFv of anti-rabbit IgG antibody (ALOB).

FIGS. 7A-D illustrate the observed frequency of occurrence of certaincombinations of CDRs in the shuffled DNA of the scFv. Panel (A) showsthe length and mutagenesis rate of all six synthetic CDRs. Panel (B)shows library construction by shuffling scFv with all six CDRs. Panel(C) shows CDR insertion determined by PCR with primers for native CDRs.Panel (D) shows insertion rates and distributions of synthetic CDRinsertions.

FIG. 8 illustrates the improved avidity of the scFv anti-rabbit antibodyafter DNA shuffling and each cycle of selection.

FIG. 9 schematically portrays pBR322-Sfi-BL-LA-Sfi and in vivointraplasmidic recombination via direct repeats, as well as the rate ofgeneration of ampicillin-resistant colonies by intraplasmidicrecombination reconstituting a functional beta-lactamase gene.

FIG. 10 schematically portrays pBR322-Sfi-2Bla-Sfi and in vivointraplasmidic recombination via direct repeats, as well as the rate ofgeneration of ampicillin-resistant colonies by intraplasmidicrecombination reconstituting a functional beta-lactamase gene.

FIGS. 11A and 11B illustrate the method for testing the efficiency ofmultiple rounds of homologous recombination after the introduction ofpolynucleotide fragments into cells for the generation of recombinantproteins.

FIG. 12 schematically portrays generation of a library of vectors byshuffling cassettes at the following loci: promoter, leader peptide,terminator, selectable drug resistance gene, and origin of replication.The multiple parallel lines at each locus represent the multiplicity ofcassettes for that cassette.

FIG. 13 schematically shows some examples of cassettes suitable atvarious loci for constructing prokaryotic vector libraries by shuffling.

FIGS. 14A and 14B show the prokaryotic GFP expression vector PBAD-GFP(5,371 bp) was derived from pBAD18 (Guzman et al. (1995) J. Bacteriol.177: 4121). The eukaryotic GFP expression vector Alpha+GFP (7,591 bp)was derived from the vector Alpha+(Whitehorn et al. (1995)Bio/Technology 13: 1215).

FIGS. 15A and 15B show comparison of the fluorescence of different GFPconstructs in whole E. Coli cells. Compared are the `Clontech` constructwhich contains a 24 amino acid N-terminal extension, the Affymaxwildtype construct (`wt`, with improved codon usage), and the mutantsobtained after 2 and after 3 cycles of sexual PCR and selection (`cycle2`, `cycle 3`). The `Clontech` construct was induced with IPTG, whereasthe other constructs were induced with arabinose. All samples wereassayed at equal OD₆₀₀. FIG. 15A shows fluorescence spectra indicatingthat the whole cell fluorescence signal from the `wt` construct is2.8-fold greater than from the `Clontech` construct. The signal of the`cycle 3` mutant is 16-fold increased over the Affymax `wt`, and 45-foldover the `Clontech` wt construct. FIG. 15B is a comparison of excitationspectra of GFP constructs in E. Coli. The peak excitation wavelengthsare unaltered by the mutations that were selected.

FIGS. 16A and 16B show SDS-PAGE analysis of relative GFP proteinexpression levels. FIG. 16A 12% Tris-Glycine SDS-PAGE analysis (Novex,Encinitas, Calif.) of equal amounts (OD600) Of whole E. coli cellsexpressing the wildtype, the cycle 2 mutant or the cycle 3 mutant ofGFP. Stained with Coomassie Blue. GFP (27 kD) represents about 75% oftotal protein, and the selection did not increase the expression level.FIG. 16B 12% Tris-Glycine SDS-PAGE analysis (Novex, Encinitas, Calif.)of equal amounts (OD600) of E. coli fractions. Lane 1: Pellet of lysedceils expressing wt GFP; lane 2: Supernatant of lysed ceils expressingwt GFP. Most of the wt GFP is in inclusion bodies; lane 3: Pellet oflysed cells expressing cycle 3 mutant GFP; lane 4: Supernatant of lysedcells expressing cycle 3 mutant GFP. Most of the wt GFPis soluble. TheGFP that ends up in inclusion bodies does not contain the chromophore,since there is no detectable fluorescence in this fraction.

FIGS. 17A and 17B show mutation analysis of the cycle 2 and cycle 3mutants versus wildtype GFP. FIG. 17A shows that the mutations arespread out rather than clustered near the tripeptide chromophore.Mutations F100S, M154T, and V164A involve the replacement of hydrophobicresidues with more hydrophilic residues. The increased hydrophilicitymay help guide the protein into a native folding pathway rather thantoward aggregation and inclusion body formation. FIG. 17B shows arestriction map indicating the chromophore region and positions ofintroduced mutations.

FIGS. 18A and 18B show comparison of CHO cells expressing different GFPproteins. FIG. 18A is a FACS analysis of clones of CHO cells expressingdifferent GFP mutants. FIG. 18B B shows fluorescence spectroscopy ofclones of CHO cells expressing different GFP mutants.

FIG. 19 shows enhancement of resistance to arsenate toxicity as a resultof shuffling the pGJ103 plasmid containing the arsenate detoxificationpathway operon.

FIG. 20 schematically shows the generation of combinatorial librariesusing synthetic or naturally-occurring intron sequences as the basis forrecombining a plurality of exons species which can lack sequenceidentity (as exemplified by random sequence exons), wherein homologousand/or site-specific recombination occurs between intron sequences ofdistinct library members.

FIGS. 21A-21G schematically show variations of the method for shufflingexons. The numbers refer to reading frames, as demonstrated in FIG. 21A.FIG. 21B shows the various classes of intron and exon relative to theirindividual splice frames. FIG. 21C provides an example of anaturally-occurring gene (immunoglobulin V genes) suitable forshuffling. FIGS. 21D through 21F shows how multiple exons can beconcatemerized via PCR using primers which span intron segments, so thatproper splicing frames are retained, if desired. FIG. 21G exemplifiesthe exon shuffling process (IG: immunoglobulin exon; IFN: interferonexon)

FIGS. 22A-22B schematically show an exon splicing frame diagram forseveral human genes, showing that preferred units for shuffling exonsbegin and end in the same splicing frame, such that a splicing module(or shuffling exon) can comprise multiple naturally-occurring exons buttypically has the same splicing frame at each end.

FIG. 23 schematically shows how partial PCR extension (stuttering) canbe used to provide recursive sequence recombination (shuffling)resulting in a library of chimeras representing multiple crossovers.

FIGS. 24A-24C show how stuttering can be used to shuffle a wild-typesequence with a multiply mutated sequence to generate an optimal set ofmutations via shuffling.

FIG. 25 schematically shows plasmid-plasmid recombination byelectroporation of a cell population representing multiple plasmidspecies, present initially as a single plasmid species per cell prior toelectroporation and multiple plasmid species per cell suitable for invivo recombination subsequent to electroporation of the cell population.

FIGS. 26A-26F show plasmid-plasmid recombination.

FIGS. 27A-27G show plasmid-virus recombination.

FIG. 28 shows virus-virus recombination.

FIGS. 29A-29E show plasmid-chromosome recombination.

FIGS. 30A-30D show conjugation-mediated recombination.

FIG. 31 shows Ab-phage recovery rate versus selection cycle. Shufflingwas applied after selection rounds two to eight. Total increase is440-fold.

FIG. 32 shows binding specificity after ten selection rounds, includingtwo rounds of backcrossing. ELISA signal of different Ab-phage clonesfor eight human protein targets.

FIGS. 33A-33B show Ab-phage recovery versus mutagenesis method.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention relates to a method for nucleic acid moleculereassembly after random fragmentation and its application to mutagenesisof DNA sequences. Also described is a method for the production ofnucleic acid fragments encoding mutant proteins having enhancedbiological activity. In particular, the present invention also relatesto a method of repeated cycles of mutagenesis, nucleic acid shufflingand selection which allow for the creation of mutant proteins havingenhanced biological activity.

The present invention is directed to a method for generating a verylarge library of DNA, RNA or protein mutants; in embodiments where ametabolic enzyme or multicomponent pathway is subjected to shuffling, alibrary can compose the resultant metabolites in addition to a libraryof the shuffled enzyme(s). This method has particular advantages in thegeneration of related DNA fragments from which the desired nucleic acidfragment(s) may be selected. In particular the present invention alsorelates to a method of repeated cycles of mutagenesis, homologousrecombination and selection which allow for the creation of mutantproteins having enhanced biological activity.

However, prior to discussing this invention in further detail, thefollowing terms will first be defined.

Definitions

As used herein, the following terms have the following meanings:

The term "DNA reassembly" is used when recombination occurs betweenidentical sequences.

By contrast, the term "DNA shuffling" is used herein to indicaterecombination between substantially homologous but non-identicalsequences, in some embodiments DNA shuffling may involve crossover vianonhomologous recombination, such as via cre/lox and/or flp/frt systemsand the like, such that recombination need not require substantiallyhomologous polynucleotide sequences. Homologous and non-homologousrecombination formats can be used, and, in some embodiments, cangenerate molecular chimeras and/or molecular hybrids of substantiallydissimilar sequences.

The term "amplification" means that the number of copies of a nucleicacid fragment is increased.

The term "identical" or "identity" means that two nucleic acid sequenceshave the same sequence or a complementary sequence. Thus, "areas ofidentity" means that regions or areas of a nucleic acid fragment orpolynucleotide are identical or complementary to another polynucleotideor nucleic acid fragment.

The term "corresponds to" is used herein to mean that a polynucleotidesequence is homologous (i.e., is identical, not strictly evolutionarilyrelated) to all or a portion of a reference polynucleotide sequence, orthat a polypeptide sequence is identical to a reference polypeptidesequence. In contradistinction, the term "complementary to" is usedherein to mean that the complementary sequence is homologous to all or aportion of a reference polynucleotide sequence. For illustration, thenucleotide sequence "TATAC" corresponds to a reference sequence "TATAC"and is complementary to a reference sequence "GTATA".

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: "reference sequence", "comparisonwindow", "sequence identity", "percentage of sequence identity", and"substantial identity". A "reference sequence" is a defined sequenceused as a basis for a sequence comparison; a reference sequence may be asubset of a larger sequence, for example, as a segment of a full-lengthcDNA or gene sequence given in a sequence listing, such as apolynucleotide sequence of FIG. 1 or FIG. 2(b), or may comprise acomplete cDNA or gene sequence. Generally, a reference sequence is atleast 20 nucleotides in length, frequently at least 25 nucleotides inlength, and often at least 50 nucleotides in length. Since twopolynucleotides may each (1) comprise a sequence (i.e., a portion of thecomplete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a "comparison window" toidentify and compare local regions of sequence similarity.

A "comparison window", as used herein, refers to a conceptual segment ofat least 20 contiguous nucleotide positions wherein a polynucleotidesequence may be compared to a reference sequence of at least 20contiguous nucleotides and wherein the portion of the polynucleotidesequence in the comparison window may comprise additions or deletions(i.e., gaps) of 20 percent or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Optimal alignment of sequences for aligning acomparison window may be conducted by the local homology algorithm ofSmith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homologyalignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988)Proc. Natl. Acad. Sci. (U.S.A.) 85: 2444, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package Release 7.0, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.), or by inspection, and the bestalignment (i.e., resulting in the highest percentage of homology overthe comparison window) generated by the various methods is selected.

The term "sequence identity" means that two polynucleotide sequences areidentical (i.e., on a nucleotide-by-nucleotide basis) over the window ofcomparison. The term "percentage of sequence identity" is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U, or I) occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison (i.e., thewindow size), and multiplying the result by 100 to yield the percentageof sequence identity. The terms "substantial identity" as used hereindenotes a characteristic of a polynucleotide sequence, wherein thepolynucleotide comprises a sequence that has at least 80 percentsequence identity, preferably at least 85 percent identity and often 90to 95 percent sequence identity, more usually at least 99 percentsequence identity as compared to a reference sequence over a comparisonwindow of at least 20 nucleotide positions, frequently over a window ofat least 25-50 nucleotides, wherein the percentage of sequence identityis calculated by comparing the reference sequence to the polynucleotidesequence which may include deletions or additions which total 20 percentor less of the reference sequence over the window of comparison.

Conservative amino acid substitutions refer to the interchangeability ofresidues having similar side chains. For example, a group of amino acidshaving aliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

The term "homologous" or "homeologous" means that one single-strandednucleic acid sequence may hybridize to a complementary single-strandednucleic acid sequence. The degree of hybridization may depend on anumber of factors including the amount of identity between the sequencesand the hybridization conditions such as temperature and saltconcentration as discussed later. Preferably the region of identity isgreater than about 5 bp, more preferably the region of identity isgreater than 10 bp.

The term "heterologous" means that one single-stranded nucleic acidsequence is unable to hybridize to another single-stranded nucleic acidsequence or its complement. Thus areas of heterology means that nucleicacid fragments or polynucleotides have areas or regions in the sequencewhich are unable to hybridize to another nucleic acid or polynucleotide.Such regions or areas are, for example, areas of mutations.

The term "cognate" as used herein refers to a gene sequence that isevolutionarily and functionally related between species. For example butnot limitation, in the human genome, the human CD4 gene is the cognategene to the mouse CD4 gene, since the sequences and structures of thesetwo genes indicate that they are highly homologous and both genes encodea protein which functions in signaling T cell activation through MHCclass II-restricted antigen recognition.

The term "wild-type" means that the nucleic acid fragment does notcomprise any mutations. A "wild-type" protein means that the proteinwill be active at a level of activity found in nature and typically willcomprise the amino acid sequence found in nature. In an aspect, the term"wild type" or "parental sequence" can indicate a starting or referencesequence prior to a manipulation of the invention.

The term "related polynucleotides" means that regions or areas of thepolynucleotides are identical and regions or areas of thepolynucleotides are heterologous.

The term "chimeric polynucleotide" means that the polynucleotidecomprises regions which are wild-type and regions which are mutated. Itmay also mean that the polynucleotide comprises wild-type regions fromone polynucleotide and wild-type regions from another relatedpolynucleotide.

The term "cleaving" means digesting the polynucleotide with enzymes orbreaking the polynucleotide, or generating partial length copies of aparent sequence(s) via partial PCR extension, PCR stuttering,differential fragment amplification, or other means of producing partiallength copies of one or more parental sequences.

The term "population" as used herein means a collection of componentssuch as polynucleotides, nucleic acid fragments or proteins. A "mixedpopulation" means a collection of components which belong to the samefamily of nucleic acids or proteins (i.e. are related) but which differin their sequence (i.e. are not identical) and hence in their biologicalactivity.

The term "specific nucleic acid fragment" means a nucleic acid fragmenthaving certain end points and having a certain nucleic acid sequence.Two nucleic acid fragments wherein one nucleic acid fragment has theidentical sequence as a portion of the second nucleic acid fragment butdifferent ends comprise two different specific nucleic acid fragments.

The term "mutations" means changes in the sequence of a wild-typenucleic acid sequence or changes in the sequence of a peptide. Suchmutations may be point mutations such as transitions or transversions.The mutations may be deletions, insertions or duplications.

In the polypeptide notation used herein, the lefthand direction is theamino terminal direction and the right-hand direction is thecarboxy-terminal direction, in accordance with standard usage andconvention. Similarly, unless specified otherwise, the lefthand end ofsingle-stranded polynucleotide sequences is the 5' end; the lefthanddirection of double-stranded polynucleotide sequences is referred to asthe 5' direction. The direction of 5' to 3' addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5' to the 5' end of the RNA transcript are referred to as "upstreamsequences"; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3' to the 3' end of the coding RNA transcriptare referred to as "downstream sequences".

The term "naturally-occurring" as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by man in the laboratory isnaturally-occurring. Generally, the term naturally-occurring refers toan object as present in a non-pathological (undiseased) individual, suchas would be typical for the species.

The term "agent" is used herein to denote a chemical compound, a mixtureof chemical compounds, an array of spatially localized compounds (e.g.,a VLSIPS peptide array, polynucleotide array, and/or combinatorial smallmolecule array), a biological macromolecule, a bacteriophage peptidedisplay library, a bacteriophage antibody (e.g., scFv) display library,a polysome peptide display library, or an extract made from biologicalmaterials such as bacteria, plants, fungi, or animal (particularlymammalian) cells or tissues. Agents are evaluated for potential activityas antineoplastics, anti-inflammatories, or apoptosis modulators byinclusion in screening assays described hereinbelow. Agents areevaluated for potential activity as specific protein interactioninhibitors (i.e., an agent which selectively inhibits a bindinginteraction between two predetermined polypeptides but which does notsubstantially interfere with cell viability) by inclusion in screeningassays described hereinbelow.

As used herein, "substantially pure" means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan any other individual macromolecular species in the composition),and preferably a substantially purified fraction is a compositionwherein the object species comprises at least about 50 percent (on amolar basis) of all macromolecular species present. Generally, asubstantially pure composition will comprise more than about 80 to 90percent of all macromolecular species present in the composition. Mostpreferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods) wherein the composition consistsessentially of a single macromolecular species. Solvent species, smallmolecules (<500 Daltons), and elemental ion species are not consideredmacromolecular species.

As used herein the term "physiological conditions" refers totemperature, pH, ionic strength, viscosity, and like biochemicalparameters which are compatible with a viable organism, and/or whichtypically exist intracellularly in a viable cultured yeast cell ormammalian cell. For example, the intracellular conditions in a yeastcell grown under typical laboratory culture conditions are physiologicalconditions. Suitable in vitro reaction conditions for in vitrotranscription cocktails are generally physiological conditions. Ingeneral, in vitro physiological conditions comprise 50-200 mM NaCl orKCl, pH 6.5-8.5, 20-45° C. and 0.001-10 mM divalent cation (e.g., Mg⁺⁺,Ca⁺⁺); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalentcation, and often include 0.01-1.0 percent nonspecific protein (e.g.,BSA). A non-ionic detergent (Tween, NP-40, Triton X-100) can often bepresent, usually at about 0.001 to 2%, typically 0.05-0.2% (v/v).Particular aqueous conditions may be selected by the practitioneraccording to conventional methods. For general guidance, the followingbuffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mMTris HCl, pH 5-8, with optional addition of divalent cation(s) and/ormetal chelators and/or nonionic detergents and/or membrane fractionsand, or antifoam agents and/or scintillants.

Specific hybridization is defined herein as the formation of hybridsbetween a first polynucleotide and a second polynucleotide (e.g., apolynucleotide having a distinct but substantially identical sequence tothe first polynucleotide), wherein the first polynucleotidepreferentially hybridizes to the second polynucleotide under stringenthybridization conditions wherein substantially unrelated polynucleotidesequences do not form hybrids in the mixture.

As used herein, the term "single-chain antibody" refers to a polypeptidecomprising a VH domain and a V_(L) domain in polypeptide linkage,generally linked via a spacer peptide (e.g., [Gly-Gly-Gly-Gly-Ser]_(x)),and which may comprise additional amino acid sequences at the amino-and/or carboxy- termini. For example, a single-chain antibody maycomprise a tether segment for linking to the encoding polynucleotide. Asan example, a scFv is a single-chain antibody. Single-chain antibodiesare generally proteins consisting of one or more polypeptide segments ofat least 10 contiguous amino acids substantially encoded by genes of theimmunoglobulin superfamily (e.g., see The Immunoglobulin GeneSuperfamily, A. F. Williams and A. N. Barclay, in Immunoglobulin Genes,T. Honjo, F. W. Alt, and T. H. Rabbitts, eds., (1989) Academic Press:San Diego, Calif., pp.361-387, which is incorporated herein byreference), most frequently encoded by a rodent, non-human primate,avian, porcine, bovine, ovine, goat, or human heavy chain or light chaingene sequence. A functional single-chain antibody generally contains asufficient portion of an immunoglobulin superfamily gene product so asto retain the property of binding to a specific target molecule,typically a receptor or antigen (epitope).

As used herein, the term "complementarity-determining region" and "CDR"refer to the art-recognized term as exemplified by the Kabat and ChothiaCDR definitions also generally known as hypervariable regions orhypervariable loops (Chothia and Lesk (1987) J. Mol. Biol. 196: 901;Chothia et al. (1989) Nature 342: 877; E. A. Kabat et al., Sequences ofProteins of Immunological Interest (National Institutes of Health,Bethesda, Md.) (1987); and Tramontano et al. (1990) J. Mol. Biol. 215:175). Variable region domains typically comprise the amino-terminalapproximately 105-115 amino acids of a naturally-occurringimmunoglobulin chain (e.g., amino acids 1-110), although variabledomains somewhat shorter or longer are also suitable for formingsingle-chain antibodies.

An immunoglobulin light or heavy chain variable region consists of a"framework" region interrupted by three hypervariable regions, alsocalled CDR's. The extent of the framework region and CDR's have beenprecisely defined (see, "Sequences of Proteins of ImmunologicalInterest," E. Kabat et al., 4th Ed., U.S. Department of Health and HumanServices, Bethesda, Md. (1987)). The sequences of the framework regionsof different light or heavy chains are relatively conserved within aspecies. As used herein, a "human framework region" is a frameworkregion that is substantially identical (about 85% or more, usually90-95% or more) to the framework region of a naturally occurring humanimmunoglobulin. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDR's. The CDR's are primarilyresponsible for binding to an epitope of an antigen.

As used herein, the term "variable segment" refers to a portion of anascent peptide which comprises a random, pseudorandom, or definedkernal sequence. A variable segment can comprise both variant andinvariant residue positions, and the degree of residue variation at avariant residue position may be limited; both options are selected atthe discretion of the practitioner. Typically, variable segments areabout 5 to 20 amino acid residues in length (e.g., 8 to 10), althoughvariable segments may be longer and may comprise antibody portions orreceptor proteins, such as an antibody fragment, a nucleic acid bindingprotein, a receptor protein, and the like.

As used herein, "random peptide sequence" refers to an amino acidsequence composed of two or more amino acid monomers and constructed bya stochastic or random process. A random peptide can include frameworkor scaffolding motifs, which may comprise invariant sequences.

As used herein "random peptide library" refers to a set ofpolynucleotide sequences that encodes a set of random peptides, and tothe set of random peptides encoded by those polynucleotide sequences, aswell as the fusion proteins containing those random peptides.

As used herein, the term "pseudorandom" refers to a set of sequencesthat have limited variability, so that for example the degree of residuevariability at one position is different than the degree of residuevariability at another position, but any pseudorandom position isallowed some degree of residue variation, however circumscribed.

As used herein, the term "defined sequence framework" refers to a set ofdefined sequences that are selected on a nonrandom basis, generally onthe basis of experimental data or structural data; for example, adefined sequence framework may comprise a set of amino acid sequencesthat are predicted to form a β-sheet structure or may comprise a leucinezipper heptad repeat motif, a zinc-finger domain, among othervariations. A "defined sequence kernal" is a set of sequences whichencompass a limited scope of variability. Whereas (1) a completelyrandom 10-mer sequence of the 20 conventional amino acids can be any of(20)¹⁰ sequences, and (2) a pseudorandom 10-mer sequence of the 20conventional amino acids can be any of (20)¹⁰ sequences but will exhibita bias for certain residues at certain positions and/or overall, (3) adefined sequence kernal is a subset of sequences which is less that themaximum number of potential sequences if each residue position wasallowed to be any of the allowable 20 conventional amino acids (and/orallowable unconventional amino/imino acids). A defined sequence kernalgenerally comprises variant and invariant residue positions and/orcomprises variant residue positions which can comprise a residueselected from a defined subset of amino acid residues), and the like,either segmentally or over the entire length of the individual selectedlibrary member sequence. Defined sequence kernals can refer to eitheramino acid sequences or polynucleotide sequences. For illustration andnot limitation, the sequences (NNK)₁₀ and (NNM)₁₀, where N represents A,T, G, or C; K represents G or T; and M represents A or C, are definedsequence kernals.

As used herein "epitope" refers to that portion of an antigen or othermacromolecule capable of forming a binding interaction that interactswith the variable region binding pocket of an antibody. Typically, suchbinding interaction is manifested as an intermolecular contact with oneor more amino acid residues of a CDR.

As used herein, "receptor" refers to a molecule that has an affinity fora given ligand. Receptors can be naturally occurring or syntheticmolecules. Receptors can be employed in an unaltered state or asaggregates with other species. Receptors can be attached, covalently ornoncovalently, to a binding member, either directly or via a specificbinding substance. Examples of receptors include, but are not limitedto, antibodies, including monoclonal antibodies and antisera reactivewith specific antigenic determinants (such as on viruses, cells, orother materials), cell membrane receptors, complex carbohydrates andglycoproteins, enzymes, and hormone receptors.

As used herein "ligand" refers to a molecule, such as a random peptideor variable segment sequence, that is recognized by a particularreceptor. As one of skill in the art will recognize, a molecule (ormacromolecular complex) can be both a receptor and a ligand. In general,the binding partner having a smaller molecular weight is referred to asthe ligand and the binding partner having a greater molecular weight isreferred to as a receptor.

As used herein, "linker" or "spacer" refers to a molecule or group ofmolecules that connects two molecules, such as a DNA binding protein anda random peptide, and serves to place the two molecules in a preferredconfiguration, e.g., so that the random peptide can bind to a receptorwith minimal steric hindrance from the DNA binding protein.

As used herein, the term "operably linked" refers to a linkage ofpolynucleotide elements in a functional relationship. A nucleic acid is"operably linked" when it is placed into a functional relationship withanother nucleic acid sequence. For instance, a promoter or enhancer isoperably linked to a coding sequence if it affects the transcription ofthe coding sequence. Operably linked means that the DNA sequences beinglinked are typically contiguous and, where necessary to join two proteincoding regions, contiguous and in reading frame.

The term "recursive sequence recombination" as used herein refers to amethod whereby a population of polynucleotide sequences are recombinedwith each other by any suitable recombination means (e.g., sexual PCR,homologous recombination, site-specific recombination, etc.) to generatea library of sequence-recombined species which is then screened orsubjected to selection to obtain those sequence-recombined specieshaving a desired property; the selected species are then subjected to atleast one additional cycle of recombination with themselves and/or withother polynucleotide species and at subsequent selection or screeningfor the desired property.

Methodology

Nucleic acid shuffling is a method for recursive in vitro or in vivohomologous recombination of pools of nucleic acid fragments orpolynucleotides. Mixtures of related nucleic acid sequences orpolynucleotides are randomly fragmented, and reassembled to yield alibrary or mixed population of recombinant nucleic acid molecules orpolynucleotides.

In contrast to cassette mutagenesis, only shuffling and error-prone PCR(or use of other mutation-enhancement methods; chemical mutagenesis,mutator strains, etc.) allow one to mutate a pool of sequences blindly(without sequence information other than primers).

The advantage of the mutagenic shuffling of this invention overerror-prone PCR alone for repeated selection can best be explained withan example from antibody engineering. In FIG. 1 is shown a schematicdiagram of DNA shuffling as described herein. The initial library canconsist of related sequences of diverse origin (i.e. antibodies fromnaive mRNA) or can be derived by any type of mutagenesis (includingshuffling) of a single antibody gene. A collection of selectedcomplementarity determining regions ("CDRs") is obtained after the firstround of affinity selection (FIG. 1). In the diagram the thick CDRsconfer onto the antibody molecule increased affinity for the antigen.Shuffling allows the free combinatorial association of all of the CDR1swith all of the CDR2s with all of the CDR3s, etc. (FIG. 1).

This method differs from PCR, in that it is an inverse chain reaction.In PCR, the number of molecules grows exponentially. In shuffling,however, the number of the polymerase start sites and the number ofmolecules remains essentially the same. When dilution is used to allowfurther lengthening of the molecules, the shuffling process becomes aninverse chain reaction generating fewer molecules.

Since cross-overs occur at regions of homology, recombination willprimarily occur between members of the same sequence family. Thisdiscourages combinations of CDRs that are grossly incompatible (eg.directed against different epitopes of the same antigen). It iscontemplated that multiple families of sequences can be shuffled in thesame reaction. Further, shuffling conserves the relative order, suchthat, for example, CDR1 will not be found in the position of CDR2.

Rare shufflants will contain a large number of the best (eg. highestaffinity) CDRs and these rare shufflants may be selected based on theirsuperior affinity (FIG. 1).

CDRs from a pool of 100 different selected antibody sequences can bepermutated in up to 100⁶ different ways. This large number ofpermutations cannot be represented in a single library of DNA sequences.Accordingly, it is contemplated that multiple cycles of DNA shufflingand selection may be required depending on the length of the sequenceand the sequence diversity desired.

Error-prone PCR, in contrast, keeps all the selected CDRs in the samerelative sequence (FIG. 1), generating a much smaller mutant cloud.

The template polynucleotide which may be used in the methods of thisinvention may be DNA or RNA. It may be of various lengths depending onthe size of the gene or DNA fragment to be recombined or reassembled.Preferably the template polynucleotide is from 50 bp to 50 kb. It iscontemplated that entire vectors containing the nucleic acid encodingthe protein of interest can be used in the methods of this invention,and in fact have been successfully used.

The template polynucleotide may be obtained by amplification using thePCR reaction (U.S. Pat. Nos. 4,683,202 and 4,683,195) or otheramplification or cloning methods. However, the removal of free primersfrom the PCR product before fragmentation provides a more efficientresult. Failure to adequately remove the primers can lead to a lowfrequency of crossover clones.

The template polynucleotide often should be double-stranded. Adouble-stranded nucleic acid molecule is required to ensure that regionsof the resulting single-stranded nucleic acid fragments arecomplementary to each other and thus can hybridize to form adouble-stranded molecule.

It is contemplated that single-stranded or double-stranded nucleic acidfragments having regions of identity to the template polynucleotide andregions of heterology to the template polynucleotide may be added to thetemplate polynucleotide at this step. It is also contemplated that twodifferent but related polynucleotide templates can be mixed at thisstep.

The double-stranded polynucleotide template and any added double-orsingle-stranded fragments are randomly digested into fragments of fromabout 5 bp to 5 kb or more. Preferably the size of the random fragmentsis from about 10 bp to 1000 bp, more preferably the size of the DNAfragments is from about 20 bp to 500 bp.

Alternatively, it is also contemplated that double-stranded nucleic acidhaving multiple nicks may be used in the methods of this invention. Anick is a break in one strand of the double-stranded nucleic acid. Thedistance between such nicks is preferably 5 bp to 5 kb, more preferablybetween 10 bp to 1000 bp.

The nucleic acid fragment may be digested by a number of differentmethods. The nucleic acid fragment may be digested with a nuclease, suchas DNAseI or RNAse. The nucleic acid may be randomly sheared by themethod of sonication or by passage through a tube having a smallorifice.

It is also contemplated that the nucleic acid may also be partiallydigested with one or more restriction enzymes, such that certain pointsof cross-over may be retained statistically.

The concentration of any one specific nucleic acid fragment will not begreater than 1% by weight of the total nucleic acid, more preferably theconcentration of any one specific nucleic acid sequence will not begreater than 0.1% by weight of the total nucleic acid.

The number of different specific nucleic acid fragments in the mixturewill be at least about 100, preferably at least about 500, and morepreferably at least about 1000.

At this step single-stranded or double-stranded nucleic acid fragments,either synthetic or natural, may be added to the random double-strandednucleic acid fragments in order to increase the heterogeneity of themixture of nucleic acid fragments.

It is also contemplated that populations of double-stranded randomlybroken or nicked nucleic acid fragments may be mixed or combined at thisstep. Damaged DNA can be exploited to enhance recombination via thenicked portions which can participate in strand invasion, formation ofrecombination junctions, serve as free 3' ends for hybrid formation andthe like.

Where insertion of mutations into the template polynucleotide isdesired, single-stranded or double-stranded nucleic acid fragmentshaving a region of identity to the template polynucleotide and a regionof heterology to the template polynucleotide may be added in a 20 foldexcess by weight as compared to the total nucleic acid, more preferablythe single-stranded nucleic acid fragments may be added in a 10 foldexcess by weight as compared to the total nucleic acid.

Where a mixture of different but related template polynucleotides isdesired, populations of nucleic acid fragments from each of thetemplates may be combined at a ratio of less than about 1:100, morepreferably the ratio is less than about 1:40. For example, a backcrossof the wild-type polynucleotide with a population of mutatedpolynucleotide may be desired to eliminate neutral mutations (e.g.,mutations yielding an insubstantial alteration in the phenotypicproperty being selected for). In such an example, the ratio of randomlydigested wild-type polynucleotide fragments which may be added to therandomly digested mutant polynucleotide fragments is approximately 1:1to about 100:1, and more preferably from 1:1 to 40:1.

The mixed population of random length nucleic acid fragments aredenatured to form single-stranded nucleic acid fragments and thenreannealed. Only those single-stranded nucleic acid fragments havingregions of homology with other single-stranded nucleic acid fragmentswill reanneal.

The random length nucleic acid fragments may be denatured by heating.One skilled in the art could determine the conditions necessary tocompletely denature the double stranded nucleic acid. Preferably thetemperature is from 80 ° C. to 100 ° C., more preferably the temperatureis from 90 ° C. to 96 ° C. Other methods which may be used to denaturethe nucleic acid fragments include pressure (36) and pH.

The nucleic acid fragments may be reannealed by cooling. Preferably thetemperature is from 20° C. to 75° C., more preferably the temperature isfrom 40° C. to 65° C. If a high frequency of crossovers is needed basedon an average of only 4 consecutive bases of homology, recombination canbe forced by using a low annealing temperature, although the processbecomes more difficult. The degree of renaturation which occurs willdepend on the degree of homology between the population ofsingle-stranded nucleic acid fragments.

Renaturation can be accelerated by the addition of polyethylene glycol("PEG") or salt. The salt concentration is preferably from 0 mM to about400 mM, more preferably the salt concentration is from 10 mM to 100 mM.The salt may be KCl or NaCl. The concentration of PEG is preferably from0% to 20%, more preferably from 5% to 10%. Higher concentrations of saltand/or PEG can be used, if desired.

The annealed nucleic acid fragments are next incubated in the presenceof a nucleic acid polymerase and dNTP's (i.e. DATP, dCTP, dGTP anddTTP). The nucleic acid polymerase may be the Klenow fragment, the Taqpolymerase or any other DNA polymerase known in the art.

The approach to be used for the assembly depends on the minimum degreeof homology that should still yield crossovers. If the areas of identityare large, Taq polymerase can be used with an annealing temperature ofbetween 45-65° C. If the areas of identity are small, Klenow polymerasecan be used with an annealing temperature of between 20-30° C. Oneskilled in the art could vary the temperature of annealing to increasethe number of cross-overs achieved.

The polymerase may be added to the random nucleic acid fragments priorto annealing, simultaneously with annealing or after annealing.

The cycle of denaturation, renaturation and incubation in the presenceof polymerase can be referred to as shuffling or reassembly of thenucleic acid. This cycle is repeated for a desired number of times.Preferably the cycle is repeated from 2 to 50 times, more preferably thesequence is repeated from 10 to 40 times. The term "shuffling"encompasses a broader range of recursive recombination processes whichcan include, but are not obligated to, PCR amplification or similaramplification methods; thus, shuffling can involve homologousrecombination, site-specific recombination, chimera formation (e.g.,Levichkin et al. op.cit), and the like, so long as used recursively(i.e., for more than one cycle of sequence recombination) with selectionand/or screening. Non-deterministic recombination, such as generalhomologous recombination can be used in combination with or in place ofdeterministic recombination, such as site-specific recombination wherethe sites of recombination are known and/or defined.

The resulting nucleic acid is a larger double-stranded polynucleotide offrom about 50 bp to about 100 kb, preferably the larger polynucleotideis from 500 bp to 50 kb.

This larger polynucleotide fragment may contain a number of copies of anucleic acid fragment having the same size as the templatepolynucleotide in tandem. This concatemeric fragment is then digestedinto single copies of the template polynucleotide. The result will be apopulation of nucleic acid fragments of approximately the same size asthe template polynucleotide. The population will be a mixed populationwhere single or double-stranded nucleic acid fragments having an area ofidentity and an area of heterology have been added to the templatepolynucleotide prior to shuffling. Alternatively, the concatemer can beintroduced (e.g., via electroporation. lipofection, or the like)directly without monomerization. For large sequences, it can bedesirable to subdivide the large sequence into several subportions whichare separately shuffled with other substantially similar portions, andthe pool of resultant shuffled subportions are then ligated, typicallyin original order, to generate a pool of shuffled large sequences whichcan then be used for transformation of a host cell, or the like.

These fragment are then cloned into the appropriate vector and theligation mixture used to transform bacteria.

It is contemplated that the single nucleic acid fragments may beobtained from the larger concatemeric nucleic acid fragment byamplification of the single nucleic acid fragments prior to cloning by avariety of methods including PCR (U.S. Pat. Nos. 4,683,195 and4,683,202) rather than by digestion of the concatemer. Alternatively,the concatemer can be introduced (e.g., via electroporation.lipofection, or the like) directly without monomerization.

The vector used for cloning is not critical provided that it will accepta DNA fragment of the desired size. If expression of the DNA fragment isdesired, the cloning vehicle should further comprise transcription andtranslation signals next to the site of insertion of the DNA fragment toallow expression of the DNA fragment in the host cell. Preferred vectorsinclude the pUC series and the pBR series of plasmids.

The resulting bacterial population will include a number of recombinantDNA fragments having random mutations. This mixed population may betested to identify the desired recombinant nucleic acid fragment. Themethod of selection will depend on the DNA fragment desired.

For example, if a DNA fragment which encodes for a protein withincreased binding efficiency to a ligand is desired, the proteinsexpressed by each of the DNA fragments in the population or library maybe tested for their ability to bind to the ligand by methods known inthe art (i.e. panning, affinity chromatography). If a DNA fragment whichencodes for a protein with increased drug resistance is desired, theproteins expressed by each of the DNA fragments in the population orlibrary may be tested for their ability to confer drug resistance to thehost organism. One skilled in the art, given knowledge of the desiredprotein, could readily test the population to identify DNA fragmentswhich confer the desired properties onto the protein.

It is contemplated that one skilled in the art could use a phage displaysystem in which fragments of the protein are expressed as fusionproteins on the phage surface (Pharmacia, Milwaukee Wis.). Therecombinant DNA molecules are cloned into the phage DNA at a site whichresults in the transcription of a fusion protein, a portion of which isencoded by the recombinant DNA molecule. The phage containing therecombinant nucleic acid molecule undergoes replication andtranscription in the cell. The leader sequence of the fusion proteindirects the transport of the fusion protein to the tip of the phageparticle. Thus the fusion protein which is partially encoded by therecombinant DNA molecule is displayed on the phage particle fordetection and selection by the methods described above.

It is further contemplated that a number of cycles of nucleic acidshuffling may be conducted with nucleic acid fragments from asubpopulation of the first population, which subpopulation contains DNAencoding the desired recombinant protein. In this manner, proteins witheven higher binding affinities or enzymatic activity could be achieved.

It is also contemplated that a number of cycles of nucleic acidshuffling may be conducted with a mixture of wild-type nucleic acidfragments and a subpopulation of nucleic acid from the first orsubsequent rounds of nucleic acid shuffling in order to remove anysilent mutations from the subpopulation.

Any source of nucleic acid, in purified form can be utilized as thestarting nucleic acid. Thus the process may employ DNA or RNA includingmessenger RNA, which DNA or RNA may be single or double stranded. Inaddition, a DNA-RNA hybrid which contains one strand of each may beutilized. The nucleic acid sequence may be of various lengths dependingon the size of the nucleic acid sequence to be mutated. Preferably thespecific nucleic acid sequence is from 50 to 50000 base pairs. It iscontemplated that entire vectors containing the nucleic acid encodingthe protein of interest may be used in the methods of this invention.

The nucleic acid may be obtained from any source, for example, fromplasmids such a pBR322, from cloned DNA or RNA or from natural DNA orRNA from any source including bacteria, yeast, viruses and higherorganisms such as plants or animals. DNA or RNA may be extracted fromblood or tissue material. The template polynucleotide may be obtained byamplification using the polynucleotide chain reaction (PCR) (U.S. Pat.Nos. 4,683,202 and 4,683,195). Alternatively, the polynucleotide may bepresent in a vector present in a cell and sufficient nucleic acid may beobtained by culturing the cell and extracting the nucleic acid from thecell by methods known in the art.

Any specific nucleic acid sequence can be used to produce the populationof mutants by the present process. It is only necessary that a smallpopulation of mutant sequences of the specific nucleic acid sequenceexist or be created prior to the present process.

The initial small population of the specific nucleic acid sequenceshaving mutations may be created by a number of different methods.Mutations may be created by error-prone PCR. Error-prone PCR useslow-fidelity polymerization conditions to introduce a low level of pointmutations randomly over a long sequence. Alternatively, mutations can beintroduced into the template polynucleotide by oligonucleotide-directedmutagenesis. In oligonucleotide-directed mutagenesis, a short sequenceof the polynucleotide is removed from the polynucleotide usingrestriction enzyme digestion and is replaced with a syntheticpolynucleotide in which various bases have been altered from theoriginal sequence. The polynucleotide sequence can also be altered bychemical mutagenesis. Chemical mutagens include, for example, sodiumbisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid. Otheragents which are analogues of nucleotide precursors includenitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Generally,these agents are added to the PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.Random mutagenesis of the polynucleotide sequence can also be achievedby irradiation with X-rays or ultraviolet light. Generally, plasmid DNAor DNA fragments so mutagenized are introduced into E. coli andpropagated as a pool or library of mutant plasmids.

Alternatively the small mixed population of specific nucleic acids maybe found in nature in that they may consist of different alleles of thesame gene or the same gene from different related species (i.e., cognategenes). Alternatively, they may be related DNA sequences found withinone species, for example, the immunoglobulin genes.

Once the mixed population of the specific nucleic acid sequences isgenerated, the polynucleotides can be used directly or inserted into anappropriate cloning vector, using techniques well-known in the art.

The choice of vector depends on the size of the polynucleotide sequenceand the host cell to be employed in the methods of this invention. Thetemplates of this invention may be plasmids, phages, cosmids, phagemids,viruses (e.g., retroviruses, parainfluenzavirus, herpesviruses,reoviruses, paramyxoviruses, and the like), or selected portions thereof(e.g., coat protein, spike glycoprotein, capsid protein). For example,cosmids, phagemids, YACs, and BACs are preferred where the specificnucleic acid sequence to be mutated is larger because these vectors areable to stably propagate large nucleic acid fragments.

If the mixed population of the specific nucleic acid sequence is clonedinto a vector it can be clonally amplified by inserting each vector intoa host cell and allowing the host cell to amplify the vector. This isreferred to as clonal amplification because while the absolute number ofnucleic acid sequences increases, the number of mutants does notincrease.

Parallel PCR

In parallel PCR a large number of different PCR reactions occur inparallel in the same vessel, with the products of one reaction primingthe products of another reaction. As the PCR products prime each other,the average product size increases with the number of PCR cycles.

By using multiple primers in parallel, sequences in excess of 50 kb canbe amplified. Whole genes and whole plasmids can be assembled in asingle tube from synthetic oligonucleotides by parallel PCR. Sequencescan be randomly mutagenized at various levels by random fragmentationand reassembly of the fragments by mutual priming. Site-specificmutations can be introduced into long sequences by random fragmentationof the template followed by reassembly of the fragments in the presenceof mutagenic oligonucleotides. A particularly useful application ofparallel PCR is called sexual PCR.

In sexual PCR, also called DNA shuffling, parallel PCR is used toperform in vitro recombination on a pool of DNA sequences. A mixture ofrelated but not identical DNA sequences (typically PCR products,restriction fragments or whole plasmids) is randomly fragmented, forexample by DNAsel treatment. These random fragments are then reassembledby parallel PCR. As the random fragments and their PCR products primeeach other, the average size of the fragments increases with the numberof PCR cycles. Recombination, or crossover, occurs by templateswitching, such as when a DNA fragment derived from one template primeson the homologous position of a related but different template. Forexample, sexual PCR can be used to construct libraries of chimaeras ofgenes from different species (`zoo libraries`). Sexual PCR is useful forin vitro evolution of DNA sequences. The libraries of new mutantcombinations that are obtained by sexual PCR are selected for the bestrecombinant sequences at the DNA, RNA, protein or small molecule level.This process of recombination, selection and amplification is repeatedfor as many cycles as necessary to obtain a desired property orfunction.

Most versions of parallel PCR do not use primers. The DNA fragments,whether synthetic, obtained by random digestion, or by PCR with primers,serve as the template as well as the primers. Because the concentrationof each different end sequence in the reassembly reaction is very low,the formation of primer dimer is not observed, and if erroneous primingoccurs, it can only grow at the same rate as the correctly annealedproduct. Parallel PCR requires many cycles of PCR because only half ofthe annealed pairs have extendable overhangs and the concentration of 3'ends is low.

Utility

The DNA shuffling method of this invention can be performed blindly on apool of unknown sequences. By adding to the reassembly mixtureoligonucleotides (with ends that are homologous to the sequences beingreassembled) any sequence mixture can be incorporated at any specificposition into another sequence mixture. Thus, it is contemplated thatmixtures of synthetic oligonucleotides, PCR fragments or even wholegenes can be mixed into another sequence library at defined positions.The insertion of one sequence (mixture) is independent from theinsertion of a sequence in another part of the template. Thus, thedegree of recombination, the homology required, and the diversity of thelibrary can be independently and simultaneously varied along the lengthof the reassembled DNA.

This approach of mixing two genes may be useful for the humanization ofantibodies from murine hybridomas. The approach of mixing two genes orinserting mutant sequences into genes may be useful for anytherapeutically used protein, for example, interleukin I, antibodies,tPA, growth hormone, etc. The approach may also be useful in any nucleicacid for example, promoters or introns or 3' untranslated region or 5'untranslated regions of genes to increase expression or alterspecificity of expression of proteins. The approach may also be used tomutate ribozymes or aptamers.

Shuffling requires the presence of homologous regions separating regionsof diversity. If the sequences to be shuffled are not substantiallyidentical, it is typically preferable to employ intron-based shufflingand/or site-specific recombination. Scaffold-like protein structures maybe particularly suitable for shuffling. The conserved scaffolddetermines the overall folding by self-association, while displayingrelatively unrestricted loops that mediate the specific binding.Examples of such scaffolds are the immunoglobulin beta-barrel, and thefour-helix bundle (24). This shuffling can be used to createscaffold-like proteins with various combinations of mutated sequencesfor binding.

In Vitro Shuffling

The equivalents of some standard genetic matings may also be performedby shuffling in vitro. For example, a `molecular backcross` can beperformed by repeated mixing of the mutant's nucleic acid with thewild-type nucleic acid while selecting for the mutations of interest. Asin traditional breeding, this approach can be used to combine phenotypesfrom different sources into a background of choice. It is useful, forexample, for the removal of neutral mutations that affect unselectedcharacteristics (i.e. immunogenicity). Thus it can be useful todetermine which mutations in a protein are involved in the enhancedbiological activity and which are not, an advantage which cannot beachieved by error-prone mutagenesis or cassette mutagenesis methods.

Large, functional genes can be assembled correctly from a mixture ofsmall random fragments. This reaction may be of use for the reassemblyof genes from the highly fragmented DNA of fossils (25). In additionrandom nucleic acid fragments from fossils may be combined with nucleicacid fragments from similar genes from related species.

It is also contemplated that the method of this invention can be usedfor the in vitro amplification of a whole genome from a single cell asis needed for a variety of research and diagnostic applications. DNAamplification by PCR is in practice limited to a length of about 40 kb.Amplification of a whole genome such as that of E. Coli (5,000 kb) byPCR would require about 250 primers yielding 125 forty kb fragments.This approach is not practical due to the unavailability of sufficientsequence data. On the other hand, random digestion of the genome withDNAseI, followed by gel purification of small fragments will provide amultitude of possible primers. Use of this mix of random small fragmentsas primers in a PCR reaction alone or with the whole genome as thetemplate should result in an inverse chain reaction with the theoreticalendpoint (assuming dilution and additional PCR) of a single concatemercontaining many copies of the genome.

100 fold amplification in the copy number and an average fragment sizeof greater than 50 kb may be obtained when only random fragments areused (see Example 2). It is thought that the larger concatemer isgenerated by overlap of many smaller fragments. The quality of specificPCR products obtained using synthetic primers will be indistinguishablefrom the product obtained from unamplified DNA. It is expected that thisapproach will be useful for the mapping of genomes.

The polynucleotide to be shuffled can be fragmented randomly ornon-randomly, at the discretion of the practitioner.

In Vivo Shuffling

In an embodiment of in vivo shuffling, the mixed population of thespecific nucleic acid sequence is introduced into bacterial (e.g.,Archeaebacteria) or eukaryotic cells under conditions such that at leasttwo different nucleic acid sequences are present in each host cell. Thefragments can be introduced into the host cells by a variety ofdifferent methods. The host cells can be transformed with the fragmentsusing methods known in the art, for example treatment with calciumchloride. If the fragments are inserted into a phage genome, the hostcell can be transfected with the recombinant phage genome having thespecific nucleic acid sequences. Alternatively, the nucleic acidsequences can be introduced into the host cell using electroporation,natural competence, transduction, transfection, lipofection, biolistics,conjugation, and the like or other suitable method of introducing apolynucleotide sequence into a cell.

In general, in this embodiment, the specific nucleic acids sequenceswill be present in vectors which are capable of stably replicating thesequence in the host cell. In addition, it is contemplated that thevectors will encode a marker gene such that host cells having the vectorcan be selected or screened. This ensures that the mutated specificnucleic acid sequence can be recovered after introduction into the hostcell. However, it is contemplated that the entire mixed population ofthe specific nucleic acid sequences need not be present on a vectorsequence. Rather only a sufficient number of sequences need be clonedinto vectors to ensure that after introduction of the fragments into thehost cells each host cell contains two vector species having at leastone related-sequence nucleic acid sequence present therein. It is alsocontemplated that rather than having a subset of the population of thespecific nucleic acids sequences cloned into vectors, this subset may bealready stably integrated into the host cell.

It has been found that when two fragments which have regions of identityare inserted into the host cells, homologous recombination occursbetween the two fragments. Such recombination between the two mutatedspecific nucleic acid sequences will result in the production ofsubstantially all combinations of all or most of the mutations (aslimited by library size and propagation efficiency, etc.).

It has also been found that the frequency of recombination is increasedif some of the mutated specific nucleic acid sequences are present onlinear nucleic acid molecules. Therefore, in a preferred embodiment,some of the specific nucleic acid sequences are present on linearnucleic acid fragments. In an embodiment, the nucleic acid molecules aresingle-stranded or substantially single-stranded, such assingle-stranded phage genomes which may or may not comprise heterologoussequences. M13 phage is an example of a suitable ssDNA template, and M13has the advantage that the M13 virus can be shuffled in prokaryoticcells (e.g., E. coli ), and then used to transfer DNA into mammaliancells.

After transformation, the host cell transformants are placed underselection to identify those host cell transformants which containmutated specific nucleic acid sequences having the qualities desired.For example, if increased resistance to a particular drug is desiredthen the transformed host cells may be subjected to increasedconcentrations of the particular drug and those transformants producingmutated proteins able to confer increased drug resistance will beselected. If the enhanced ability of a particular protein to bind to areceptor is desired, then expression of the protein can be induced fromthe transformants and the resulting protein assayed in a ligand bindingassay by methods known in the art to identify that subset of the mutatedpopulation which shows enhanced binding to the ligand. Alternatively,the protein can be expressed in another system to ensure properprocessing.

Once a subset of the first recombined specific nucleic acid sequences(daughter sequences) having the desired characteristics are identified,they are then subject to a second round of recombination.

In the second cycle of recombination, the recombined specific nucleicacid sequences may be mixed with the original mutated specific nucleicacid sequences (parent sequences) and the cycle repeated as describedabove. In this way a set of second recombined specific nucleic acidssequences can be identified which have enhanced characteristics orencode for proteins having enhanced properties. This cycle can berepeated a number of times as desired.

It is also contemplated that in the second or subsequent recombinationcycle, a backcross can be performed. A molecular backcross can beperformed by mixing the desired specific nucleic acid sequences with alarge number of the wild-type sequence, such that at least one wild-typenucleic acid sequence and a mutated nucleic acid sequence are present inthe same host cell after transformation. Recombination with thewild-type specific nucleic acid sequence will eliminate those neutral orweakly contributory mutations that may affect unselected characteristicssuch as immunogenicity but not the selected characteristics.

In another embodiment of this invention, it is contemplated that duringthe first round a subset of the specific nucleic acid sequences can befragmented prior to introduction into the host cell. The size of thefragments must be large enough to contain some regions of identity withthe other sequences so as to homologously recombine with the othersequences. These fragments, ssDNA or dsDNA, can be coated with RecA invitro to promote hybridization and/or integration into the host DNA. Thesize of the fragments will range from 0.03 kb to 100 kb more preferablyfrom 0.2 kb to 10 kb. It is also contemplated that in subsequent rounds,all of the specific nucleic acid sequences other than the sequencesselected from the previous round may be cleaved into fragments prior tointroduction into the host cells. "Cleavage" may be by nucleasedigestion, PCR amplification (via partial extension or stuttering), orother suitable means for generating partial length polynucleotides ofparent sequence(s).

Fragmentation of the sequences can be accomplished by a variety ofmethods known in the art. The sequences can be randomly fragmented orfragmented at specific sites in the nucleic acid sequence. Randomfragments can be obtained by breaking the nucleic acid or exposing it toharsh physical treatment (e.g., shearing or irradiation) or harshchemical agents (e.g., by free radicals; metal ions; acid treatment todepurinate and cleave). Random fragments can also be obtained, in thecase of DNA by the use of DNase or like nuclease, or by other means asdiscussed herein. The sequences can be cleaved at specific sites by theuse of restriction enzymes. The fragmented sequences can besingle-stranded or double-stranded. If the sequences were originallysingle-stranded they can be denatured with heat, chemicals or enzymesprior to insertion into the host cell. The reaction conditions suitablefor separating the strands of nucleic acid are well known in the art.Furthermore, partial PCR extension, PCR stuttering, and other relatedmethods for producing partial length copies of a parental sequence canbe used to effect "fragmentation", e.g., to obtain a hybrid productwhich contains segments derived from different parental sequences.

The steps of this process can be repeated indefinitely, being limitedonly by the number of possible mutants which can be achieved. After acertain number of cycles, all possible mutants will have been achievedand further cycles are redundant.

In an embodiment the same mutated template nucleic acid is repeatedlyrecombined and the resulting recombinants selected for the desiredcharacteristic.

Therefore, the initial pool or population of mutated template nucleicacid is cloned into a vector capable of replicating in bacteria such asE. coli . The particular vector is not essential, so long as it iscapable of autonomous replication in E. coli or integration into a hostchromosome. In a preferred embodiment, the vector is designed to allowthe expression and production of any protein encoded by the mutatedspecific nucleic acid linked to the vector. It is also preferred thatthe vector contain a gene encoding for a selectable marker.

The population of vectors containing the pool of mutated nucleic acidsequences is introduced into the E. coli host cells. The vector nucleicacid sequences may be introduced by transformation, transfection orinfection in the case of phage. The concentration of vectors used totransform the bacteria is such that a number of vectors is introducedinto each cell. Once present in the cell, the efficiency of homologousrecombination is such that homologous recombination occurs between thevarious vectors. This results in the generation of mutants (daughters)having a combination of mutations which differ from the original parentmutated sequences.

The host cells are then replicated, typically clonally, and selected forthe marker gene present on the vector. Only those cells having a plasmidwill grow under the selection.

The host cells which contain a vector are then tested for the presenceof favorable mutations. Such testing may consist of placing the cellsunder selective pressure, for example, if the gene to be selected is animproved drug resistance gene. If the vector allows expression of theprotein encoded by the mutated nucleic acid sequence, then suchselection may include allowing expression of the protein so encoded,isolation of the protein and testing of the protein to determinewhether, for example, it binds with increased efficiency to the ligandof interest.

Once a particular pool of daughter mutated nucleic acid sequence hasbeen identified which confers the desired characteristics, the nucleicacid is isolated either already linked to the vector or separated fromthe vector. This nucleic acid is then recombined with itself or withsimilarly selected pools) and the cycle is repeated; optionally parentalsequences can be used for subsequent rounds of recombination, in placeof or in addition to other selected daughter species.

It has been shown that by this method nucleic acid sequences havingenhanced desired properties can be selected.

In an alternate embodiment, the first generation of mutants are retainedin the cells and the first generation of mutant sequences are addedagain to the cells. Accordingly, the first cycle of Embodiment I isconducted as described above. However, after the daughter nucleic acidsequences are identified, the host cells containing these sequences areretained.

The daughter mutated specific nucleic acid population, either asfragments or cloned into the same vector is introduced into the hostcells already containing the daughter nucleic acids. Recombination isallowed to occur in the cells and the next generation of recombinants,or granddaughters are selected by the methods described above.

This cycle can be repeated a number of times until the nucleic acid orpeptide having the desired characteristics is obtained. It iscontemplated that in subsequent cycles, the population of mutatedsequences which are added to the preferred mutants may come from theparental mutants or any subsequent generation.

In an alternative embodiment, the invention provides a method ofconducting a "molecular" backcross of the obtained recombinant specificnucleic acids (one species or a pool of several species) in order toeliminate any neutral mutations. Neutral mutations are those mutationswhich do not confer onto the nucleic acid or peptide the desiredproperties. Such mutations may however confer on the nucleic acid orpeptide undesirable characteristics. Accordingly, it is desirable toeliminate such neutral mutations. The methods of this invention providea means of doing so.

In this embodiment, after the mutant nucleic acid, having the desiredcharacteristics, is obtained by the methods of the embodiments, thenucleic acid, the vector having the nucleic acid or the host cell,tissue, or individual organism containing the vector and nucleic acid isisolated.

The nucleic acid or vector is then introduced into the host cell with alarge excess of the wild-type nucleic acid. The nucleic acid of themutant and the nucleic acid of the wild-type sequence are allowed torecombine. The resulting recombinants are placed under the sameselection as the mutant nucleic acid. Only those recombinants whichretained the desired characteristics will be selected. Any silentmutations which do not provide the desired characteristics will be lostthrough recombination with the wild-type DNA. This cycle can be repeateda number of times until all of the silent mutations are eliminated.

Thus the methods of this invention can be used in a molecular backcrossto eliminate unnecessary, weakly contributing, and/or silent mutations.

Utility

The in vivo recombination method of this invention can be performedblindly on a pool of unknown mutants or alleles of a specific nucleicacid fragment or sequence, or family of diverse but related sequences orsequences which share one or more recombinogenic sequences (e.g., asite-specific recombination site, a localized segment of sequencehomology having substantial identity for homologous recombination, ahomologous recombination "hotspot" sequence, a restriction site, etc.)suitable for recursive recombination. However, it is not necessary toknow the actual DNA or RNA sequence of the specific nucleic acidfragment.

The approach of using recombination within a mixed population of genescan be useful for the generation of any useful proteins, for example,interleukin I, antibodies, tPA, growth hormone, etc. This approach maybe used to generate proteins having altered specificity or activity. Theapproach may also be useful for the generation of mutant nucleic acidsequences, for example, promoter regions, introns, exons, enhancersequences, 3' untranslated regions or 5' untranslated regions of genes.Thus this approach may be used to generate genes having increased ratesof expression. This approach may also be useful in the study ofrepetitive DNA sequences. Finally, this approach may be useful to mutateribozymes or aptamers.

Scaffold-like regions separating regions of diversity in proteins may beparticularly suitable for the methods of this invention. The conservedscaffold determines the overall folding by self-association, whiledisplaying relatively unrestricted loops that mediate the specificbinding. Examples of such scaffolds are the immunoglobulin beta barrel,and the four-helix bundle. The methods of this invention can be used tocreate scaffold-like proteins with various combinations of mutatedsequences for binding.

The equivalents of some standard genetic matings may also be performedby the methods of this invention. For example, a "molecular" backcrosscan be performed by repeated mixing of the mutant's nucleic acid withthe wild-type nucleic acid while selecting for the mutations ofinterest. As in traditional breeding, this approach can be used tocombine phenotypes from different sources into a background of choice.It is useful, for example, for the removal of neutral mutations thataffect unselected characteristics (i.e. immunogenicity). Thus it can beuseful to determine which mutations in a protein are involved in theenhanced biological activity and which are not.

Peptide Display Methods

The present method can be used to shuffle, by in vitro and/or in vivorecombination by any of the disclosed methods, and in any combination,polynucleotide sequences selected by peptide display methods, wherein anassociated polynucleotide encodes a displayed peptide which is screenedfor a phenotype (e.g., for affinity for a predetermined receptor(ligand).

An increasingly important aspect of biopharmaceutical drug developmentand molecular biology is the identification of peptide structures,including the primary amino acid sequences, of peptides orpeptidomimetics that interact with biological macromolecules. One methodof identifying peptides that possess a desired structure or functionalproperty, such as binding to a predetermined biological macromolecule(e.g., a receptor), involves the screening of a large library orpeptides for individual library members which possess the desiredstructure or functional property conferred by the amino acid sequence ofthe peptide.

In addition to direct chemical synthesis methods for generating peptidelibraries, several recombinant DNA methods also have been reported. Onetype involves the display of a peptide sequence, antibody, or otherprotein on the surface of a bacteriophage particle or cell. Generally,in these methods each bacteriophage particle or cell serves as anindividual library member displaying a single species of displayedpeptide in addition to the natural bacteriophage or cell proteinsequences. Each bacteriophage or cell contains the nucleotide sequenceinformation encoding the particular displayed peptide sequence; thus,the displayed peptide sequence can be ascertained by nucleotide sequencedetermination of an isolated library member.

A well-known peptide display method involves the presentation of apeptide sequence on the surface of a filamentous bacteriophage,typically as a fusion with a bacteriophage coat protein. Thebacteriophage library can be incubated with an immobilized,predetermined macromolecule or small molecule (e.g., a receptor) so thatbacteriophage particles which present a peptide sequence that binds tothe immobilized macromolecule can be differentially partitioned fromthose that do not present peptide sequences that bind to thepredetermined macromolecule. The bacteriophage particles (i.e., librarymembers) which are bound to the immobilized macromolecule are thenrecovered and replicated to amplify the selected bacteriophagesubpopulation for a subsequent round of affinity enrichment and phagereplication. After several rounds of affinity enrichment and phagereplication, the bacteriophage library members that are thus selectedare isolated and the nucleotide sequence encoding the displayed peptidesequence is determined, thereby identifying the sequence(s) of peptidesthat bind to the predetermined macromolecule (e.g., receptor). Suchmethods are further described in PCT patent publication Nos. 91/17271,91/18980, and 91/19818 and 93/08278.

The latter PCT publication describes a recombinant DNA method for thedisplay of peptide ligands that involves the production of a library offusion proteins with each fusion protein composed of a first polypeptideportion, typically comprising a variable sequence, that is available forpotential binding to a predetermined macromolecule, and a secondpolypeptide portion that binds to DNA, such as the DNA vector encodingthe individual fusion protein. When transformed host cells are culturedunder conditions that allow for expression of the fusion protein, thefusion protein binds to the DNA vector encoding it. Upon lysis of thehost cell, the fusion protein/vector DNA complexes can be screenedagainst a predetermined macromolecule in much the same way asbacteriophage particles are screened in the phage-based display system,with the replication and sequencing of the DNA vectors in the selectedfusion protein/vector DNA complexes serving as the basis foridentification of the selected library peptide sequence(s).

Other systems for generating libraries of peptides and like polymershave aspects of both the recombinant and in vitro chemical synthesismethods. In these hybrid methods, cell-free enzymatic machinery isemployed to accomplish the in vitro synthesis of the library members(i.e., peptides or polynucleotides). In one type of method, RNAmolecules with the ability to bind a predetermined protein or apredetermined dye molecule were selected by alternate rounds ofselection and PCR amplification (Tuerk and Gold (1990) Science 249: 505;Ellington and Szostak (1990) Nature 346: 818). A similar technique wasused to identify DNA sequences which bind a predetermined humantranscription factor (Thiesen and Bach (1990) Nucleic Acids Res. 18:3203; Beaudry and Joyce (1992) Science 257; 635; PCT patent publicationNos. 92/05258 and 92/14843). In a similar fashion, the technique of invitro translation has been used to synthesize proteins of interest andhas been proposed as a method for generating large libraries ofpeptides. These methods which rely upon in vitro translation, generallycomprising stabilized polysome complexes, are described further in PCTpatent publication Nos. 88/08453, 90/05785, 90/07003, 91/02076,91/05058, and 92/02536. Applicants have described methods in whichlibrary members comprise a fusion protein having a first polypeptideportion with DNA binding activity and a second polypeptide portionhaving the library member unique peptide sequence; such methods aresuitable for use in cell-free in vitro selection formats, among others.

A variation of the method is recursive sequence recombination performedby intron-based recombination, wherein the sequences to be recombinedare present as exons (e.g., in the form of exons, whether naturallyoccurring or artificial) which may share substantial, little, or nosequence identity and which are separated by one or more introns (whichmay be naturally occurring intronic sequences or not) which sharesufficient sequence identity to support homologous recombination betweenintrons. For example but not limitation, a population of polynucleotidescomprises library members wherein each library member has one or morecopies of a first set of exons linked via a first set of introns to oneor more copies of a second set of exons linked via a second set ofintrons to one or more copies of a third set of exons. Each of themembers of the first set of exons may share substantial, little, or nosequence identity with each other or with members of the second or thirdsets of exons. Similarly, each of the members of the second set of exonsmay share substantial, little, or no sequence identity with each otheror with members of the first or third sets of exons. Similarly, each ofthe members of the third set of exons may share substantial, little, orno sequence identity with each other or with members of the first orsecond sets of exons. Each of the members of the each set (first,second, third, etc.) of introns shares sufficient sequence identity withthe other members of the set to support recombination (homologous orsite-specific recombination, including restriction site-mediatedrecombination) between members of the same intron set, but typically notwith members of other intron sets (e.g., the second or third sets), suchthat intra-set recombination between introns of the library membersoccurs and generates a pool of recombined library members wherein thefirst set of exons, second set of exons, and third set of exons areeffectively shuffled with each other.

The displayed peptide sequences can be of varying lengths, typicallyfrom 3-5000 amino acids long or longer, frequently from 5-100 aminoacids long, and often from about 8-15 amino acids long. A library cancomprise library members having varying lengths of displayed peptidesequence, or may comprise library members having a fixed length ofdisplayed peptide sequence. Portions or all of the displayed peptidesequence(s) can be random, pseudorandom, defined set kernal, fixed, orthe like. The present display methods include methods for in vitro andin vivo display of single-chain antibodies, such as nascent scFv onpolysomes or scFv displayed on phage, which enable large-scale screeningof scFv libraries having broad diversity of variable region sequencesand binding specificities.

The present invention also provides random, pseudorandom, and definedsequence framework peptide libraries and methods for generating andscreening those libraries to identify useful compounds (e.g., peptides,including single-chain antibodies) that bind to receptor molecules orepitopes of interest or gene products that modify peptides or RNA in adesired fashion. The random, pseudorandom, and defined sequenceframework peptides are produced from libraries of peptide librarymembers that comprise displayed peptides or displayed single-chainantibodies attached to a polynucleotide template from which thedisplayed peptide was synthesized. The mode of attachment may varyaccording to the specific embodiment of the invention selected, and caninclude encapsidation in a phage particle or incorporation in a cell.

A method of affinity enrichment allows a very large library of peptidesand single-chain antibodies to be screened and the polynucleotidesequence encoding the desired peptide(s) or single-chain antibodies tobe selected. The pool of polynucleotides can then be isolated andshuffled to recombine combinatorially the amino acid sequence of theselected peptide(s) (or predetermined portions thereof) or single-chainantibodies (or just V_(H), V_(L), or CDR portions thereof). Using thesemethods, one can identify a peptide or single-chain antibody as having adesired binding affinity for a molecule and can exploit the process ofshuffling to converge rapidly to a desired high-affinity peptide orscFv. The peptide or antibody can then be synthesized in bulk byconventional means for any suitable use (e.g., as a therapeutic ordiagnostic agent).

A significant advantage of the present invention is that no priorinformation regarding an expected ligand structure is required toisolate peptide ligands or antibodies of interest. The peptideidentified can have biological activity, which is meant to include atleast specific binding affinity for a selected receptor molecule and, insome instances, will further include the ability to block the binding ofother compounds, to stimulate or inhibit metabolic pathways, to act as asignal or messenger, to stimulate or inhibit cellular activity, and thelike.

The present invention also provides a method for shuffling a pool ofpolynucleotide sequences selected by affinity screening a library ofpolysomes displaying nascent peptides (including single-chainantibodies) for library members which bind to a predetermined receptor(e.g., a mammalian proteinaceous receptor such as, for example, apeptidergic hormone receptor, a cell surface receptor, an intracellularprotein which binds to other protein(s) to form intracellular proteincomplexes such as heterodimers and the like) or epitope (e.g., animmobilized protein, glycoprotein, oligosaccharide, and the like).

Polynucleotide sequences selected in a first selection round (typicallyby affinity selection for binding to a receptor (e.g., a ligand) by anyof these methods are pooled and the pool(s) is/are shuffled by in vitroand/or in vivo recombination to produce a shuffled pool comprising apopulation of recombined selected polynucleotide sequences. Therecombined selected polynucleotide sequences are subjected to at leastone subsequent selection round. The polynucleotide sequences selected inthe subsequent selection round(s) can be used directly (as a pool or inindividual clones), sequenced, and/or subjected to one or moreadditional rounds of shuffling and subsequent selection. Selectedsequences can also be backcrossed with polynucleotide sequences encodingneutral sequences (i.e., having insubstantial functional effect onbinding), such as for example by backcrossing with a wild-type ornaturally-occurring sequence substantially identical to a selectedsequence to produce native-like functional peptides, which may be lessimmunogenic. Generally, during backcrossing subsequent selection isapplied to retain the property of binding (or other desired selectableor screenable property or phenotype) to the predetermined receptor(ligand). Other properties are exemplified by the capacity to block apredetermined binding interaction (e.g., act as a partial or completeantagonist or competitive binding species) or to exhibit a catalyticfunction, or the like, among others.

Prior to or concomitant with the shuffling of selected sequences, thesequences can be mutagenized. In one embodiment, selected librarymembers are cloned in a prokaryotic vector (e.g., plasmid, phagemid, orbacteriophage) wherein a collection of individual colonies (or plaques)representing discrete library members are produced. Individual selectedlibrary members can then be manipulated (e.g., by site-directedmutagenesis, cassette mutagenesis, chemical mutagenesis, PCRmutagenesis, and the like) to generate a collection of library membersrepresenting a kernal of sequence diversity based on the sequence of theselected library member. The sequence of an individual selected librarymember or pool can be manipulated to incorporate random mutation,pseudorandom mutation, defined kernal mutation (i.e., comprising variantand invariant residue positions and/or comprising variant residuepositions which can comprise a residue selected from a defined subset ofamino acid residues), codon-based mutation, and the like, eithersegmentally or over the entire length of the individual selected librarymember sequence. The mutagenized selected library members are thenshuffled by in vitro and/or in vivo recombinatorial shuffling asdisclosed herein.

The invention also provides peptide libraries comprising a plurality ofindividual library members of the invention, wherein (1) each individuallibrary member of said plurality comprises a sequence produced byshuffling of a pool of selected sequences, and (2) each individuallibrary member comprises a variable peptide segment sequence orsingle-chain antibody segment sequence which is distinct from thevariable peptide segment sequences or single-chain antibody sequences ofother individual library members in said plurality (although somelibrary members may be present in more than one copy per library due touneven amplification, stochastic probability, or the like).

The invention also provides a product-by-process, wherein selectedpolynucleotide sequences having (or encoding a peptide having) apredetermined binding specificity are formed by the process of: (1)screening a displayed peptide or displayed single-chain antibody libraryagainst a predetermined receptor (e.g., ligand) or epitope (e.g.,antigen macromolecule) and identifying and/or enriching library memberswhich bind to the predetermined receptor or epitope to produce a pool ofselected library members, (2) shuffling by recombination of the selectedlibrary members (or amplified or cloned copies thereof) which binds thepredetermined epitope and has been thereby isolated and/or enriched fromthe library to generate a shuffled library, and (3) screening theshuffled library against the predetermined receptor (e.g., ligand) orepitope (e.g., antigen macromolecule) and identifying and/or enrichingshuffled library members which bind to the predetermined receptor orepitope to produce a pool of selected shuffled library members.

Antibody Display and Screening Methods

The present method can be used to shuffle, by in vitro and/or in vivorecombination by any of the disclosed methods, and in any combination,polynucleotide sequences selected by antibody display methods, whereinan associated polynucleotide encodes a displayed antibody which isscreened for a phenotype (e.g., for affinity for binding a predeterminedantigen (ligand).

Various molecular genetic approaches have been devised to capture thevast immunological repertoire represented by the extremely large numberof distinct variable regions which can be present in immunoglobulinchains. The naturally-occurring germline immunoglobulin heavy chainlocus is composed of separate tandem arrays of variable (V) segmentgenes located upstream of a tandem array of diversity (D) segment genes,which are themselves located upstream of a tandem array of joining (J)region genes, which are located upstream of the constant (C_(H)) regiongenes. During B lymphocyte development, V-D-J rearrangement occurswherein a heavy chain variable region gene (V_(H)) is formed byrearrangement to form a fused D-J segment followed by rearrangement witha V segment to form a V-D-J joined product gene which, if productivelyrearranged, encodes a functional variable region (V_(H)) of a heavychain. Similarly, light chain loci rearrange one of several V segmentswith one of several J segments to form a gene encoding the variableregion (V_(L)) of a light chain.

The vast repertoire of variable regions possible in immunoglobulinsderives in part from the numerous combinatorial possibilities of joiningV and J segments (and, in the case of heavy chain loci, D segments)during rearrangement in B cell development. Additionalsequence-diversity in the heavy chain variable regions arises fromnon-uniform rearrangements of the D segments during V-D-J joining andfrom N region addition. Further, antigen-selection of specific B cellclones selects for higher affinity variants having nongermline mutationsin one or both of the heavy and light chain variable regions; aphenomenon referred to as "affinity maturation" or "affinitysharpening". Typically, these "affinity sharpening" mutations cluster inspecific areas of the variable region, most commonly in thecomplementarity-determining regions (CDRs).

In order to overcome many of the limitations in producing andidentifying high-affinity immunoglobulins through antigen-stimulated Bcell development (i.e., immunization), various prokaryotic expressionsystems have been developed that can be manipulated to producecombinatorial antibody libraries which may be screened for high-affinityantibodies to specific antigens. Recent advances in the expression ofantibodies in Escherichia coli and bacteriophage systems (see,"Alternative Peptide Display Methods", infra) have raised thepossibility that virtually any specificity can be obtained by eithercloning antibody genes from characterized hybridomas or by de novoselection using antibody gene libraries (e.g., from Ig cDNA).

Combinatorial libraries of antibodies have been generated inbacteriophage lambda expression systems which may be screened asbacteriophage plaques or as colonies of lysogens (Huse et al. (1989)Science 246: 1275; Caton and Koprowski (1990) Proc. Natl. Acad. Sci.(U.S.A.) 87: 6450; Mullinax et al (1990) Proc. Natl. Acad. Sci. (U.S.A.)87: 8095; Persson et al. (1991) Proc. Natl. Acad. Sci. (U.S.A.) 88:2432). Various embodiments of bacteriophage antibody display librariesand lambda phage expression libraries have been described (Kang et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 4363; Clackson et al. (1991)Nature 352: 624; McCafferty et al. (1990) Nature 348: 552; Burton et al.(1991) Proc. Natl. Acad. Sci. (U.S.A.) 88: 10134; Hoogenboom et al.(1991) Nucleic Acids Res. 19: 4133; Chang et al. (1991) J. Immunol. 147:3610; Breitling et al. (1991) Gene 104: 147; Marks et al. (1991) J. Mol.Biol. 222: 581; Barbas et al. (1992) Proc. Natl. Acad. Sci. (U.S.A.) 89:4457; Hawkins and Winter (1992) J. Immunol. 22: 867; Marks et al. (1992)Biotechnology 10: 779; Marks et al. (1992) J. Biol. Chem. 267: 16007;Lowman et al (1991) Biochemistry 30: 10832; Lerner et al. (1992) Science258: 1313, incorporated herein by reference). Typically, a bacteriophageantibody display library is screened with a receptor (e.g., polypeptide,carbohydrate, glycoprotein, nucleic acid) that is immobilized (e.g., bycovalent linkage to a chromatography resin to enrich for reactive phageby affinity chromatography) and/or labeled (e.g., to screen plaque orcolony lifts).

One particularly advantageous approach has been the use of so-calledsingle-chain fragment variable (scFv) libraries (Marks et al. (1992)Biotechnology 10: 779; Winter G and Milstein C (1991) Nature 349: 293;Clackson et al. (1991) op.cit.; Marks et al. (1991) J. Mol. Biol. 222:581; Chaudhary et al. (1990) Proc. Natl. Acad. Sci. (USA) 87: 1066;Chiswell et al. (1992) TIBTECH 10: 80; McCafferty et al. (1990) op.cit.;and Huston et al. (1988) Proc. Natl. Acad. Sci. (USA) 85: 5879). Variousembodiments of scFv libraries displayed on bacteriophage coat proteinshave been described.

Beginning in 1988, single-chain analogues of Fv fragments and theirfusion proteins have been reliably generated by antibody engineeringmethods. The first step generally involves obtaining the genes encodingV_(H) and V_(L) domains with desired binding properties; these V genesmay be isolated from a specific hybridoma cell line, selected from acombinatorial V-gene library, or made by V gene synthesis. Thesingle-chain Fv is formed by connecting the component V genes with anoligonucleotide that encodes an appropriately designed linker peptide,such as (Gly-Gly-Gly-Gly-Ser)₃ or equivalent linker peptide(s). Thelinker bridges the C-terminus of the first V region and N-terminus ofthe second, ordered as either VH₃ -linker-V_(L) or V_(L) -linker-V_(H).In principle, the scFv binding site can faithfully replicate both theaffinity and specificity of its parent antibody combining site.

Thus, scFv fragments are comprised of V_(H) and V_(L) domains linkedinto a single polypeptide chain by a flexible linker peptide. After thescFv genes are assembled, they are cloned into a phagemid and expressedat the tip of the M13 phage (or similar filamentous bacteriophage) asfusion proteins with the bacteriophage pIII (gene 3) coat protein.Enriching for phage expressing an antibody of interest is accomplishedby panning the recombinant phage displaying a population scFv forbinding to a predetermined epitope (e.g., target antigen, receptor).

The linked polynucleotide of a library member provides the basis forreplication of the library member after a screening or selectionprocedure, and also provides the basis for the determination, bynucleotide sequencing, of the identity of the displayed peptide sequenceor V_(H) and V_(L) amino acid sequence. The displayed peptide(s) orsingle-chain antibody (e.g., scFv) and/or its V_(H) and V_(L) domains ortheir CDRs can be cloned and expressed in a suitable expression system.Often polynucleotides encoding the isolated V_(H) and V_(L) domains willbe ligated to polynucleotides encoding constant regions (C_(H) andC_(L)) to form polynucleotides encoding complete antibodies (e.g.,chimeric or fully-human), antibody fragments, and the like. Oftenpolynucleotides encoding the isolated CDRs will be grafted intopolynucleotides encoding a suitable variable region framework (andoptionally constant regions) to form polynucleotides encoding completeantibodies (e.g., humanized or fully-human), antibody fragments, and thelike. Antibodies can be used to isolate preparative quantities of theantigen by immunoaffinity chromatography. Various other uses of suchantibodies are to diagnose and/or stage disease (e.g., neoplasia), andfor therapeutic application to treat disease, such as for example:neoplasia, autoimmune disease, AIDS, cardiovascular disease, infections,and the like.

Various methods have been reported for increasing the combinatorialdiversity of a scFv library to broaden the repertoire of binding species(idiotype spectrum). The use of PCR has permitted the variable regionsto be rapidly cloned either from a specific hybridoma source or as agene library from non-immunized cells, affording combinatorial diversityin the assortment of V_(H) and V_(L) cassettes which can be combined.Furthermore, the V_(H) and V_(L) cassettes can themselves bediversified, such as by random, pseudorandom, or directed mutagenesis.Typically, V_(H) and V_(L) cassettes are diversified in or near thecomplementarity-determining regions (CDRs), often the third CDR, CDR3.Enzymatic inverse PCR mutagenesis has been shown to be a simple andreliable method for constructing relatively large libraries of scFvsite-directed mutants (Stemmer et al. (1993) Biotechniques 14: 256), ashas error-prone PCR and chemical mutagenesis (Deng et al. (1994) J.Biol. Chem. 269: 9533). Riechmann et al. (1993) Biochemistry 32: 8848showed semirational design of an antibody scFv fragment usingsite-directed randomization by degenerate oligonucleotide PCR andsubsequent phage display of the resultant scFv mutants. Barbas et al.(1992) op.cit. attempted to circumvent the problem of limited repertoiresizes resulting from using biased variable region sequences byrandomizing the sequence in a synthetic CDR region of a human tetanustoxoid-binding Fab.

CDR randomization has the potential to create approximately 1×10²⁰ CDRsfor the heavy chain CDR3 alone, and a roughly similar number of variantsof the heavy chain CDR1 and CDR2, and light chain CDR1-3 variants. Takenindividually or together, the combinatorics of CDR randomization ofheavy and/or light chains requires generating a prohibitive number ofbacteriophage clones to produce a clone library representing allpossible combinations, the vast majority of which will be non-binding.Generation of such large numbers of primary transformants is notfeasible with current transformation technology and bacteriophagedisplay systems. For example, Barbas et al. (1992) op.cit. onlygenerated 5×10⁷ transformants, which represents only a tiny fraction ofthe potential diversity of a library of thoroughly randomized CDRs.

Despite these substantial limitations, bacteriophage display of scFv hasalready yielded a variety of useful antibodies and antibody fusionproteins. A bispecific single chain antibody has been shown to mediateefficient tumor cell lysis (Gruber et al. (1994) J. Immunol. 152: 5368).Intracellular expression of an anti-Rev scFv has been shown to inhibitHIV-1 virus replication in vitro (Duan et al. (1994) Proc. Natl. Acad.Sci. (USA) 91: 5075), and intracellular expression of an anti-p21^(ras)scFv has been shown to inhibit meiotic maturation of Xenopus oocytes(Biocca et al. (1993) Biochem. Biophys. Res. Commun. 197: 422.Recombinant scFv which can be used to diagnose HIV infection have alsobeen reported, demonstrating the diagnostic utility of scFv (Lilley etal. (1994) J. Immunol. Meth. 171: 211). Fusion proteins wherein an scFvis linked to a second polypeptide, such as a toxin or fibrinolyticactivator protein, have also been reported (Holvost et al. (1992) Eur.J. Biochem. 210: 945; Nicholls et al. (1993) J. Biol. Chem. 268: 5302).

If it were possible to generate scFv libraries having broader antibodydiversity and overcoming many of the limitations of conventional CDRmutagenesis and randomization methods which can cover only a very tinyfraction of the potential sequence combinations, the number and qualityof scFv antibodies suitable for therapeutic and diagnostic use could bevastly improved. To address this, the in vitro and in vivo shufflingmethods of the invention are used to recombine CDRs which have beenobtained (typically via PCR amplification or cloning) from nucleic acidsobtained from selected displayed antibodies. Such displayed antibodiescan be displayed on cells, on bacteriophage particles, on polysomes, orany suitable antibody display system wherein the antibody is associatedwith its encoding nucleic acid(s). In a variation, the CDRs areinitially obtained from mRNA (or cDNA) from antibody-producing cells(e.g., plasma cells/splenocytes from an immunized wild-type mouse, ahuman, or a transgenic mouse capable of making a human antibody as inW092/03918, W093/12227, and W094/25585), including hybridomas derivedtherefrom.

Polynucleotide sequences selected in a first selection round (typicallyby affinity selection for displayed antibody binding to an antigen(e.g., a ligand) by any of these methods are pooled and the pool(s)is/are shuffled by in vitro and/or in vivo recombination, especiallyshuffling of CDRs (typically shuffling heavy chain CDRs with other heavychain CDRs and light chain CDRs with other light chain CDRs) to producea shuffled pool comprising a population of recombined selectedpolynucleotide sequences. The recombined selected polynucleotidesequences are expressed in a selection format as a displayed antibodyand subjected to at least one subsequent selection round. Thepolynucleotide sequences selected in the subsequent selection round(s)can be used directly, sequenced, and/or subjected to one or moreadditional rounds of shuffling and subsequent selection until anantibody of the desired binding affinity is obtained. Selected sequencescan also be backcrossed with polynucleotide sequences encoding neutralantibody framework sequences (i.e., having insubstantial functionaleffect on antigen binding), such as for example by backcrossing with ahuman variable region framework to produce human-like sequenceantibodies. Generally, during backcrossing subsequent selection isapplied to retain the property of binding to the predetermined antigen.

Alternatively, or in combination with the noted variations, the valencyof the target epitope may be varied to control the average bindingaffinity of selected scFv library members. The target epitope can bebound to a surface or substrate at varying densities, such as byincluding a competitor epitope, by dilution, or by other method known tothose in the art. A high density (valency) of predetermined epitope canbe used to enrich for scFv library members which have relatively lowaffinity, whereas a low density (valency) can preferentially enrich forhigher affinity scFv library members.

For generating diverse variable segments, a collection of syntheticoligonucleotides encoding random, pseudorandom, or a defined sequencekernal set of peptide sequences can be inserted by ligation into apredetermined site (e.g., a CDR). Similarly, the sequence diversity ofone or more CDRs of the single-chain antibody cassette(s) can beexpanded by mutating the CDR(s) with site-directed mutagenesis,CDR-replacement, and the like. The resultant DNA molecules can bepropagated in a host for cloning and amplification prior to shuffling,or can be used directly (i.e., may avoid loss of diversity which mayoccur upon propagation in a host cell) and the selected library memberssubsequently shuffled.

Displayed peptide/polynucleotide complexes (library members) whichencode a variable segment peptide sequence of interest or a single-chainantibody of interest are selected from the library by an affinityenrichment technique. This is accomplished by means of a immobilizedmacromolecule or epitope specific for the peptide sequence of interest,such as a receptor, other macromolecule, or other epitope species.Repeating the affinity selection procedure provides an enrichment oflibrary members encoding the desired sequences, which may then beisolated for pooling and shuffling, for sequencing, and/or for furtherpropagation and affinity enrichment.

The library members without the desired specificity are removed bywashing. The degree and stringency of washing required will bedetermined for each peptide sequence or single-chain antibody ofinterest and the immobilized predetermined macromolecule or epitope. Acertain degree of control can be exerted over the bindingcharacteristics of the nascent peptide/DNA complexes recovered byadjusting the conditions of the binding incubation and the subsequentwashing. The temperature, pH, ionic strength, divalent cationsconcentration, and the volume and duration of the washing will selectfor nascent peptide/DNA complexes within particular ranges of affinityfor the immobilized macromolecule. Selection based on slow dissociationrate, which is usually predictive of high affinity, is often the mostpractical route. This may be done either by continued incubation in thepresence of a saturating amount of free predetermined macromolecule, orby increasing the volume, number, and length of the washes. In eachcase, the rebinding of dissociated nascent peptide/DNA or peptide/RNAcomplex is prevented, and with increasing time, nascent peptide/DNA orpeptide/RNA complexes of higher and higher affinity are recovered.

Additional modifications of the binding and washing procedures may beapplied to find peptides with special characteristics. The affinities ofsome peptides are dependent on ionic strength or cation concentration.This is a useful characteristic for peptides that will be used inaffinity purification of various proteins when gentle conditions forremoving the protein from the peptides are required.

One variation involves the use of multiple binding targets (multipleepitope species, multiple receptor species), such that a scFv librarycan be simultaneously screened for a multiplicity of scFv which havedifferent binding specificities. Given that the size of a scFv libraryoften limits the diversity of potential scFv sequences, it is typicallydesirable to use scFv libraries of as large a size as possible. The timeand economic considerations of generating a number of very largepolysome scFv-display libraries can become prohibitive. To avoid thissubstantial problem, multiple predetermined epitope species (receptorspecies) can be concomitantly screened in a single library, orsequential screening against a number of epitope species can be used. Inone variation, multiple target epitope species, each encoded on aseparate bead (or subset of beads), can be mixed and incubated with apolysome-display scFv library under suitable binding conditions. Thecollection of beads, comprising multiple epitope species, can then beused to isolate, by affinity selection, scFv library members. Generally,subsequent affinity screening rounds can include the same mixture ofbeads, subsets thereof, or beads containing only one or two individualepitope species. This approach affords efficient screening, and iscompatible with laboratory automation, batch processing, and highthroughput screening methods.

A variety of techniques can be used in the present invention todiversify a peptide library or single-chain antibody library, or todiversify, prior to or concomitant with shuffling, around variablesegment peptides or V_(H), V_(L), or CDRs found in early rounds ofpanning to have sufficient binding activity to the predeterminedmacromolecule or epitope. In one approach, the positive selectedpeptide/polynucleotide complexes (those identified in an early round ofaffinity enrichment) are sequenced to determine the identity of theactive peptides. Oligonucleotides are then synthesized based on theseactive peptide sequences, employing a low level of all basesincorporated at each step to produce slight variations of the primaryoligonucleotide sequences. This mixture of (slightly) degenerateoligonucleotides is then cloned into the variable segment sequences atthe appropriate locations. This method produces systematic, controlledvariations of the starting peptide sequences, which can then beshuffled. It requires, however, that individual positive nascentpeptide/polynucleotide complexes be sequenced before mutagenesis, andthus is useful for expanding the diversity of small numbers of recoveredcomplexes and selecting variants having higher binding affinity and/orhigher binding specificity. In a variation, mutagenic PCR amplificationof positive selected peptide/polynucleotide complexes (especially of thevariable region sequences, the amplification products of which areshuffled in vitro and/or in vivo and one or more additional rounds ofscreening is done prior to sequencing. The same general approach can beemployed with single-chain antibodies in order to expand the diversityand enhance the binding affinity/specificity, typically by diversifyingCDRs or adjacent framework regions prior to or concomitant withshuffling. If desired, shuffling reactions can be spiked with mutagenicoligonucleotides capable of in vitro recombination with the selectedlibrary members. Thus, mixtures of synthetic oligonucleotides and PCRfragments (synthesized by error-prone or high-fidelity methods) can beadded to the in vitro shuffling mix and be incorporated into resultingshuffled library members (shufflants).

The present invention of shuffling enables the generation of a vastlibrary of CDR-variant single-chain antibodies. One way to generate suchantibodies is to insert synthetic CDRs into the single-chain antibodyand/or CDR randomization prior to or concomitant with shuffling. Thesequences of the synthetic CDR cassettes are selected by referring toknown sequence data of human CDR and are selected in the discretion ofthe practitioner according to the following guidelines: synthetic CDRswill have at least 40 percent positional sequence identity to known CDRsequences, and preferably will have at least 50 to 70 percent positionalsequence identity to known CDR sequences. For example, a collection ofsynthetic CDR sequences can be generated by synthesizing a collection ofoligonucleotide sequences on the basis of naturally-occurring human CDRsequences listed in Kabat et al. (1991) o. cit.; the pool(s) ofsynthetic CDR sequences are calculated to encode CDR peptide sequenceshaving at least 40 percent sequence identity to at least one knownnaturally-occurring human CDR sequence. Alternatively, a collection ofnaturally-occurring CDR sequences may be compared to generate consensussequences so that amino acids used at a residue position frequently(i.e., in at least 5 percent of known CDR sequences) are incorporatedinto the synthetic CDRs at the corresponding position(s). Typically,several (e.g., 3 to about 50) known CDR sequences are compared andobserved natural sequence variations between the known CDRs aretabulated, and a collection of oligonucleotides encoding CDR peptidesequences encompassing all or most permutations of the observed naturalsequence variations is synthesized. For example but not for limitation,if a collection of human V_(H) CDR sequences have carboxy-terminal aminoacids which are either Tyr, Val, Phe, or Asp, then the pool(s) ofsynthetic CDR oligonucleotide sequences are designed to allow thecarboxy-terminal CDR residue to be any of these amino acids. In someembodiments, residues other than those which naturally-occur at aresidue position in the collection of CDR sequences are incorporated:conservative amino acid substitutions are frequently incorporated and upto 5 residue positions may be varied to incorporate non-conservativeamino acid substitutions as compared to known naturally-occurring CDRsequences. Such CDR sequences can be used in primary library members(prior to first round screening) and/or can be used to spike in vitroshuffling reactions of selected library member sequences. Constructionof such pools of defined and/or degenerate sequences will be readilyaccomplished by those of ordinary skill in the art.

The collection of synthetic CDR sequences comprises at least one memberthat is not known to be a naturally-occurring CDR sequence. It is withinthe discretion of the practitioner to include or not include a portionof random or pseudorandom sequence corresponding to N region addition inthe heavy chain CDR; the N region sequence ranges from 1 nucleotide toabout 4 nucleotides occurring at V-D and D-J junctions. A collection ofsynthetic heavy chain CDR sequences comprises at least about 100 uniqueCDR sequences, typically at least about 1,000 unique CDR sequences,preferably at least about 10,000 unique CDR sequences, frequently morethan 50,000 unique CDR sequences; however, usually not more than about1×10⁶ unique CDR sequences are included in the collection, althoughoccasionally 1×10⁷ to 1×10⁸ unique CDR sequences are present, especiallyif conservative amino acid substitutions are permitted at positionswhere the conservative amino acid substituent is not present or is rare(i.e., less than 0.1 percent) in that position in naturally-occurringhuman CDRs. In general, the number of unique CDR sequences included in alibrary generally should not exceed the expected number of primarytransformants in the library by more than a factor of 10. Suchsingle-chain antibodies generally bind to a predetermined antigen (e.g.,the immunogen) with an affinity of about at least 1×10⁷ M⁻¹, preferablywith an affinity of about at least 5×10⁷ M⁻¹, more preferably with anaffinity of at least 1×10⁸ M⁻¹ to 1×10⁹ M⁻¹ or more, sometimes up to1×10¹⁰ M⁻¹ or more. Frequently, the predetermined antigen is a humanprotein, such as for example a human cell surface antigen (e.g. , CD4,CD8, IL-2 receptor, EGF receptor, PDGF receptor), other human biologicalmacromolecule (e.g., thrombomodulin,l protein C, carbohydrate antigen,sialyl Lewis antigen, L-selectin), or nonhuman disease associatedmacromolecule (e.g. , bacterial LPS, virion capsid protein or envelopeglycoprotein) and the like.

High affinity single-chain antibodies of the desired specificity can beengineered and expressed in a variety of systems. For example, scFv havebeen produced in plants (Firek et al. (1993) Plant Mol. Biol. 23: 861)and can be readily made in prokaryotic systems (Owens RJ and Young RJ(1994) J. Immunol. Meth. 168: 149; Johnson S and Bird RE (1991) MethodsEnzymol. 203: 88). Furthermore, the single-chain antibodies can be usedas a basis for constructing whole antibodies or various fragmentsthereof (Kettleborough et al. (1994) Eur. J. Immunol. 24: 952). Thevariable region encoding sequence may be isolated (e.g., by PCRamplification or subcloning) and spliced to a sequence encoding adesired human constant region to encode a human sequence antibody moresuitable f or human therapeutic uses where immunogenicity is preferablyminimized. The polynucleotide(s) having the resultant fully humanencoding sequence (s) can be expressed in a host cell (e.g., from anexpression vector in a mammalian cell) and purified for pharmaceuticalformulation.

The DNA expression constructs will typically include an expressioncontrol DNA sequence operably linked to the coding sequences, includingnaturally-associated or heterologous promoter regions. Preferably, theexpression control sequences will be eukaryotic promoter systems invectors capable of transforming or transfecting eukaryotic host cells.Once the vector has been incorporated into the appropriate host, thehost is maintained under conditions suitable for high level expressionof the nucleotide sequences, and the collection and purification of themutant "engineered" antibodies.

As stated previously, the DNA sequences will be expressed in hosts afterthe sequences have been operably linked to an expression controlsequence (i.e., positioned to ensure the transcription and translationof the structural gene). These expression vectors are typicallyreplicable in the host organisms either as episomes or as an integralpart of the host chromosomal DNA. Commonly, expression vectors willcontain selection markers, e.g., tetracycline or neomycin, to permitdetection of those cells transformed with the desired DNA sequences(see, e.g., U.S. Pat. No. 4,704,362, which is incorporated herein byreference).

In addition to eukaryotic microorganisms such as yeast, mammalian tissuecell culture may also be used to produce the polypeptides of the presentinvention (see, Winnacker, "From Genes to Clones," VCH Publishers, N.Y.,N.Y. (1987), which is incorporated herein by reference). Eukaryoticcells are actually preferred, because a number of suitable host celllines capable of secreting intact immunoglobulins have been developed inthe art, and include the CHO cell lines, various COS cell lines, HeLacells, myeloma cell lines, etc, but preferably transformed B-cells orhybridomas. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer (Queen et al. (1986) Immunol. Rev. 89: 49), and necessaryprocessing information sites, such as ribosome binding sites, RNA splicesites, polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromimmunoglobulin genes, cytomegalovirus, SV40, Adenovirus, BovinePapilloma Virus, and the like.

Eukaryotic DNA transcription can be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting sequences of between10 to 300 bp that increase transcription by a promoter. Enhancers caneffectively increase transcription when either 5' or 3' to thetranscription unit. They are also effective if located within an intronor within the coding sequence itself. Typically, viral enhancers areused, including SV40 enhancers, cytomegalovirus enhancers, polyomaenhancers, and adenovirus enhancers. Enhancer sequences from mammaliansystems are also commonly used, such as the mouse immunoglobulin heavychain enhancer.

Mammalian expression vector systems will also typically include aselectable marker gene. Examples of suitable markers include, thedihydrofolate reductase gene (DHFR), the thymidine kinase gene (TK), orprokaryotic genes conferring drug resistance. The first two marker genesprefer the use of mutant cell lines that lack the ability to growwithout the addition of thymidine to the growth medium. Transformedcells can then be identified by their ability to grow onnon-supplemented media. Examples of prokaryotic drug resistance genesuseful as markers include genes conferring resistance to G418,mycophenolic acid and hygromycin.

The vectors containing the DNA segments of interest can be transferredinto the host cell by well-known methods, depending on the type ofcellular host. For example, calcium chloride transfection is commonlyutilized for prokaryotic cells, whereas calcium phosphate treatment.lipofection, or electroporation may be used for other cellular hosts.Other methods used to transform mammalian cells include the use ofPolybrene, protoplast fusion, liposomes, electroporation, andmicroinjection (see, generally, Sambrook et al., supra).

Once expressed, the antibodies, individual mutated immunoglobulinchains, mutated antibody fragments, and other immunoglobulinpolypeptides of the invention can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,fraction column chromatography, gel electrophoresis and the like (see,generally, Scopes, R., Protein Purification, Springer-Verlag, N.Y.(1982)). Once purified, partially or to homogeneity as desired, thepolypeptides may then be used therapeutically or in developing andperforming assay procedures, immunofluorescent stainings, and the like(see, generally, Immunological Methods, Vols. I and II, Eds. Lefkovitsand Pernis, Academic Press, New York, N.Y. (1979 and 1981)).

The antibodies generated by the method of the present invention can beused for diagnosis and therapy. By way of illustration and notlimitation, they can be used to treat cancer, autoimmune diseases, orviral infections. For treatment of cancer, the antibodies will typicallybind to an antigen expressed preferentially on cancer cells, such aserbB-2, CEA, CD33, and many other antigens and binding members wellknown to those skilled in the art.

Yeast Two-Hybrid Screening Assays

Shuffling can also be used to recombinatorially diversify a pool ofselected library members obtained by screening a two-hybrid screeningsystem to identify library members which bind a predeterminedpolypeptide sequence. The selected library members are pooled andshuffled by in vitro and/or in vivo recombination. The shuffled pool canthen be screened in a yeast two hybrid system to select library memberswhich bind said predetermined polypeptide sequence (e.g., and SH2domain) or which bind an alternate predetermined polypeptide sequence(e.g., an SH2 domain from another protein species).

An approach to identifying polypeptide sequences which bind to apredetermined polypeptide sequence has been to use a so-called"two-hybrid" system wherein the predetermined polypeptide sequence ispresent in a fusion protein (Chien et al. (1991) Proc. Natl. Acad. Sci.(USA) 88: 9578). This approach identifies protein-protein interactionsin vivo through reconstitution of a transcriptional activator (Fields Sand Song (1989) Nature 340: 245), the yeast Gal4 transcription protein.Typically, the method is based on the properties of the yeast Gal4protein, which consists of separable domains responsible for DNA-bindingand transcriptional activation. Polynucleotides encoding two hybridproteins, one consisting of the yeast Gal4 DNA-binding domain fused to apolypeptide sequence of a known protein and the other consisting of theGal4 activation domain fused to a polypeptide sequence of a secondprotein, are constructed and introduced into a yeast host cell.Intermolecular binding between the two fusion proteins reconstitutes theGal4 DNA-binding domain with the Ga14 activation domain, which leads tothe transcriptional activation of a reporter gene (e.g., lacZ, HIS3)which is operably linked to a Gal4 binding site. Typically, thetwo-hybrid method is used to identify novel polypeptide sequences whichinteract with a known protein (Silver SC and Hunt SW (1993) Mol. Biol.Rep. 17: 155; Durfee et al. (1993) Genes Devel. 7; 555; Yang et al.(1992) Science 257: 680; Luban et al. (1993) Cell 73: 1067; Hardy et al.(1992) Genes Devel. 6; 801; Bartel et al. (1993) Biotechniques 14: 920;and Vojtek et al. (1993) Cell 74: 205). However, variations of thetwo-hybrid method have been used to identify mutations of a knownprotein that affect its binding to a second known protein (Li B andFields S (1993) FASEB J. 7: 957; Lalo et al. (1993) Proc. Natl. Acad.Sci. (USA) 90: 5524; Jackson et al. (1993) Mol. Cell. Biol. 13; 2899;and Madura et al. (1993) J. Biol. Chem. 268: 12046). Two-hybrid systemshave also been used to identify interacting structural domains of twoknown proteins (Bardwell et al. (1993) med. Microbiol. 8: 1177;Chakraborty et al. (1992) J. Biol. Chem. 267: 17498; Staudinger et al.(1993) J. Biol. Chem. 268: 4608; and Milne GT and Weaver DT (1993) GenesDevel. 7; 1755) or domains responsible for oligomerization of a singleprotein (Iwabuchi et al. (1993) Oncogene 8; 1693; Bogerd et al. (1993)J. Virol. 67: 5030). Variations of two-hybrid systems have been used tostudy the in vivo activity of a proteolytic enzyme (Dasmahapatra et al.(1992) Proc. Natl. Acad. Sci. (USA) 89: 4159). Alternatively, an E. coli/BCCP interactive screening system (Germino et al. (1993) Proc. Natl.Acad. Sci. (U.S.A.) 90: 933; Guarente L (1993) Proc. Natl. Acad. Sci.(U.S.A.) 90: 1639) can be used to identify interacting protein sequences(i.e., protein sequences which heterodimerize or form higher orderheteromultimers) . Sequences selected by a two-hybrid system can bepooled and shuffled and introduced into a two-hybrid system for one ormore subsequent rounds of screening to identify polypeptide sequenceswhich bind to the hybrid containing the predetermined binding sequence.The sequences thus identified can be compared to identify consensussequence(s) and consensus sequence kernals.

Improvements/Alternative Formats

Additives

In one aspect, the improved shuffling method includes the addition of atleast one additive which enhances the rate or extent of reannealing orrecombination of related-sequence polynucleotides. In general, additiveswhich increase hybrid stability of mismatched sequences can be used toenhance the frequency of generating substantially mutated librarymembers (i.e., having a greater mutational density). In addition toadditives, modulation of the ionic strength (e.g., Na⁺ and/or K⁺ ionconcentration) can modulate the relative stability of mismatchedhybrids, such that increased salt concentration can increase thefrequency of mismatched hybrids and contribute to formation of librarymembers having multiple mutations.

In an embodiment, the additive is polyethylene glycol (PEG), typicallyadded to a shuffling reaction to a final concentration of 0.1 to 25percent, often to a final concentration of 2.5 to 15 percent, to a finalconcentration of about 10 percent. In an embodiment, the additive isdextran sulfate, typically added to a shuffling reaction to a finalconcentration of 0.1 to 25 percent, often at about 10 percent. In anembodiment, the additive is an agent which reduces sequence specificityof reannealing and promotes promiscuous hybridization and/orrecombination in vitro. In an alternative embodiment, the additive is anagent which increases sequence specificity of reannealing and promoteshigh fidelity hybridization and/or recombination in vitro. Otherlong-chain polymers which do not interfere with the reaction may also beused (e.g., polyvinylpyrrolidone, etc.).

In one aspect, the improved shuffling method includes the addition of atleast one additive which is a cationic detergent. Examples of suitablecationic detergents include but are not limited to:cetyltrimethylammonium bromide (CTAB), dodecyltrimethylammonium bromide(DTAB) , and tetramethylammonium chloride (TMAC), and the like.

In one aspect, the improved shuffling method includes the addition of atleast one additive which is a recombinogenic protein that catalzyes ornon-catalytically enhances homologous pairing and/or strand exchange invitro. Examples of suitable recombinogenic proteins include but are notlimited to: E. coli recA protein, the T4 uvsX protein, the redl proteinfrom Ustilago maydis, other recA family recombinases from other species,single strand binding protein (SSB), ribonucleoprotein A1, and the like.Nucleases and proofreading polymerases are often included to improve themaintenance of 3' end integrity. Each of these protein additives canthemselves be improved by multiple rounds of recursive sequencerecombination and selection and/or screening. The invention embracessuch improved additives and their use to further enhance shuffling.

Recombinase Proteins

Recombinases are proteins that, when included with an exogenoustargeting polynucleotide, provide a measurable increase in therecombination frequency and/or localization frequency between thetargeting polynucleotide and an endogenous predetermined DNA sequence.In the present invention, recombinase refers to a family of RecA-likerecombination proteins all having essentially all or most of the samefunctions, particularly: (i) the recombinase protein's ability toproperly bind to and position targeting polynucleotides on theirhomologous targets and (ii) the ability of recombinase protein/targetingpolynucleotide complexes to efficiently find and bind to complementaryendogenous sequences. The best characterized recA protein is from E.coli , in addition to the wild-type protein a number of mutant recA-likeproteins have been identified (e.g., recA803). Further, many organismshave recA-like recombinases with strand-transfer activities (e.g.,Fugisawa et al., (1985) Nucl. Acids Res. 13: 7473; Hsieh et al., (1986)Cell 44: 885; Hsieh et al., (1989) J. Biol. Chem. 264: 5089; Fishel etal., (1988) Proc. Natl. Acad. Sci. USA 85: 3683; Cassuto et al., (1987)Mol. Gen. Genet. 208: 10; Ganea et al., (1987) Mol. Cell Biol. 7: 3124;Moore et al., (1990) J. Biol. Chem. 19: 11108; Keene et al., (1984)Nucl. Acids Res. 12: 3057; Kimiec, (1984) Cold Spring Harbor Symp.48:675; Kimeic, (1986) Cell 44: 545; Kolodner et al., (1987) Proc. Natl.Acad. Sci. USA 84 :5560; Sugino et al., (1985) Proc. Natl. Acad. Sci.USA 85: 3683; Halbrook et al., (1989) J. Biol. Chem. 264: 21403; Eisenet al., (1988) Proc. Natl. Acad. Sci. USA 85: 7481; McCarthy et al.,(1988) Proc. Natl. Acad. Sci. USA 85: 5854; Lowenhaupt et al., (1989) J.Biol. Chem. 264: 20568, which are incorporated herein by reference.Examples of such recombinase proteins include, for example but notlimitation: recA, recA803, uvsX, and other recA mutants and recA-likerecombinases (Roca, A. I. (1990) Crit. Rev. Biochem. Molec. Biol. 25:415), sepl (Kolodner et al. (1987) Proc. Natl. Acad. Sci. (U.S.A.) 84:5560; Tishkoff et al. Molec. Cell. Biol. 11: 2593), RuvC (Dunderdale etal. (1991) Nature 354: 506), DST2, KEM1, XRN1 (Dykstra et al. (1991)Molec. Cell. Biol. 11: 2583), STPa/DST1 (Clark et al. (1991) Molec.Cell. Biol. 11: 2576), HPP-1 (Moore et al. (1991) Proc. Natl. Acad. Sci.(U.S.A.) 88: 9067), other eukaryotic recombinases (Bishop et al. (1992)Cell 69: 439; Shinohara et al. (1992) Cell 69: 457); incorporated hereinby reference. RecA may be purified from E. coil strains, such as E. colistrains JC12772 and JC15369 (available from A. J. Clark and M. Madiraju,University of California-Berkeley). These strains contain the recAcoding sequences on a "runaway" replicating plasmid vector present at ahigh copy number per cell. The recA803 protein is a high-activity mutantof wild-type recA. The art teaches several examples of recombinaseproteins, for example, from Drosophila, yeast, plant, human, andnon-human mammalian cells, including proteins with biological propertiessimilar to recA (i.e., recA-like recombinases).

RecA protein is typically obtained from bacterial strains thatoverproduce the protein: wild-type E. coli recA protein and mutantrecA803 protein may be purified from such strains. Alternatively, recAprotein can also be purchased from, for example, Pharmacia (Piscataway,N.J.).

RecA protein forms a nucleoprotein filament when it coats asingle-stranded DNA. In this nucleoprotein filament, one monomer of recAprotein is bound to about 3 nucleotides. This property of recA to coatsingle-stranded DNA is essentially sequence independent, althoughparticular sequences favor initial loading of recA onto a polynucleotide(e.g., nucleation sequences). The nucleoprotein filament(s) can beformed on essentially any sequence-related polypeptide to be suffled andcan be formed in cells (e.g., bacterial, yeast, or mammalian cells),forming complexes with both single-stranded and double-stranded DNA.

Site-specific recombination can be used to accomplish recursive sequencerecombination. Typically, sequences to be shuffled are flanked by one ormore site-specific recombination sequences, such as for example a FLPrecombination target site (FRT) often consisting of the two inverted 13base repeats and an 8 bp spacer (O'Gorman et al. (1991) Science 251:1351; Parsons et al. (1990) J. Biol. Chem. 265: 4527; Amin et al. (1991)Mol. Cell. Biol. 11: 4497, incorporated herein by reference). When FRTsequences are employed, the FLP recombinase is typically also employed,either in vitro or expressed in a host cell wherein the sequences to berecombined are introduced or are already present. Alternatives to theFLP/FRT system include, but are not limited to, the cre/lox system ofphage P1 (Hoess and Abremski (1985) J. Mol. Biol. 181: 351), the γ/δresolvase (Steitz et al. (1990) Quarterly Rev. Biophys. 23: 205), theattB/attP system of λ (Nunes-Duby et al. (1987) Cell 50: 779), and likesite-specific recombination systems from bacteriophages λ, φ80, P22, P2,P4, P1, and other like site-specific recombination systems selected bythe practioner. Guidance regarding the integrase family of recombinasesis found in Argos et al. (1986) EMBO J. 5: 433, incorporated herein byreference.

Exonuclease

In one aspect, the improved shuffling method includes the addition of atleast one additive which is an enzyme having an exonuclease activitywhich is active at removing non-templated nucleotides introduced at 3'ends of product polynucleotides in shuffling amplification reactionscatalyzed by a non-proofreading polymerase. An examples of a suitableenzyme having an exonuclease activity includes but is not limited to Pfupolymerase. Examples of exonucleases are:

Bal31

Bacteriophage Lambda exonuclease

E. coli Exonuclease I

E. coli Exonuclease III

E. coli Exonuclease VII

Bacteriophage T7 gene 6.

Stuttering

In an aspect, the improved shuffling method comprises the modificationwherein at least one cycle of amplification (i.e., extension with apolymerase) of reannealed fragmented library member polynucleotides isconducted under conditions which produce a substantial fraction,typically at least 20 percent or more, of incompletely extendedamplification products. The amplification products, including theincompletely extended amplification products are denatured and subjectedto at least one additional cycle of reannealing and amplification. Thisvariation, wherein at least one cycle of reannealing and amplificationprovides a substantial fraction of incompletely extended products, istermed "stuttering" and in the subsequent amplification round theincompletely extended products reanneal to and prime extension ondifferent sequence-related template species.

In an aspect, the improved shuffling method comprises the modificationwherein at least one cycle of amplification is conducted using acollection of overlapping single-stranded DNA fragments of varyinglengths corresponding to a first polynucleotide species or set ofrelated-sequence polynucleotide species, wherein each overlappingfragment can each hybridize to and prime polynucleotide chain extensionfrom a second polynucleotide species serving as a template, thus formingsequence-recombined polynucleotides, wherein said sequence-recombinedpolynucleotides comprise a portion of at least one first polynucleotidespecies with an adjacent portion of the second polynucleotide specieswhich serves as a template. In a variation, the second polynucleotidespecies serving as a template contains uracil (i.e., a Kunkel-typetemplate) and is substantially non-replicable in cells. This aspect ofthe invention can also comprise at least two recursive cycles of thisvariation. In one embodiment, recursive cycles of shuffling using themethod of Levitchkin et al. (1995) Mol. Biol. 29: 572, which producespartial extension PCR fragments is used to generate chimeras from a poolof parental sequences which are recursively shuffled.

In an aspect, the improved shuffling method comprises the modificationwherein at least one cycle of amplification is conducted with anadditive or polymerase in suitable conditions which promote templateswitching. In an embodiment where Taq polymerase is employed foramplification, addition of recA or other polymerases enhances templateswitching. Template-switching can also be increased by increasing theDNA template concentration, among other means known by those skilled inthe art.

In an embodiment of the general method, libraries of sequence-recombinedpolynucleotides are generated from sequence-related polynucleotideswhich are naturally-occurring genes or alleles of a gene. In thisaspect, at least three naturally-occurring genes and/or alleles whichcomprise regions of at least 50 consecutive nucleotides which have atleast 70 percent sequence identity, prefereably at least 90 percentsequence identity, are selected from a pool of gene sequences, such asby hybrid selection or via computerized sequence analysis using sequencedata from a database. The selected sequences are obtained aspolynucleotides, either by cloning or via DNA synthesis, and shuffled byany of the various embodiments of the invention.

In an embodiment of the invention, the method comprises the further stepof removing non-shuffled products (e.g., parental sequences) fromsequence-recombined polynucleotides produced by any of the disclosedshuffling methods. Non-shuffled products can be removed or avoided byperforming amplification with: (1) a first PCR primer which hybridizesto a first parental polynucleotide species but does not substantiallyhybridize to a second parental polynucleotide species, and (2) a secondPCR primer which hybridizes to a second parental polynucleotide speciesbut does not substantially hybridize to the first parentalpolynucleotide species, such that amplification occurs on from templatescomprising the portion of the first parental sequence which hybridizesto the first PCR primer and also comprising the portion of the secondparental sequence which hybridizes to the second PCR primer, thus onlysequence-recombined polynucleotides are amplified.

In an embodiment of the invention, "bridging" genes can be synthesized.If two or more parental polynucelotides (e.g., genes) lack satisfactorysequence similarity for efficient homologous recombination or forefficient cross-priming for PCR amplification, intermediate (or"bridging") genes can be synthesized which share sufficient sequenceidentity with the parental sequences. The bridging gene need not beactive or confer a phenotype or selectable property, it need onlyprovide a template having sufficient sequence identity to accomplishshuffling of the parental sequences. The intermediate homology of thebridging gene, and the necessary sequencers) can be determined bycomputer or manually.

The invention also provides additional formats for performing recursiverecombination in vivo, either in procaryotic or eucaryotic cells. Theseformats include recombination between plasmids, recombination betweenviruses, recombination between plasmid and virus, recombination betweena chromosome and plasmid or virus and intramolecular recombination(e.g., between two sequences on a plasmid). Recursive recombination canbe performed entirely in vivo whereby successive rounds of in vivorecombination are interspersed by rounds of selection or screening. Invivo formats can also be used in combination with in vitro formats. Forexample, one can perform one round of in vitro shuffling, a round ofselection, a round of in vivo shuffling, a further round of selection, afurther round of in vitro shuffling and a further round of selection andso forth. The various in vivo formats are now considered in turn.

(a) Plasmid-Plasmid Recombination

The initial substrates for recombination are a collection ofpolynucleotides comprising variant forms of a gene. The variant formsusually show substantial sequence identity to each other sufficient toallow homologous recombination between substrates. The diversity betweenthe polynucleotides can be natural (e.g., allelic or species variants),induced (e.g., error-prone PCR, synthetic genes, codon-usage alteredsequence variants), or the result of in vitro recombination. Thereshould be at least sufficient diversity between substrates thatrecombination can generate more diverse products than there are startingmaterials. There must be at least two substrates differing in at leasttwo positions. However, commonly a library of substrates of 10³ -10⁸members is employed. The degree of diversity depends on the length ofthe substrate being recombined and the extent of the functional changeto be evolved. Diversity at between 0.1-25% of positions is typical.

The diverse substrates are incorporated into plasmids. The plasmids areoften standard cloning vectors, e.g., bacterial multicopy plasmids.However, in some methods to be described below, the plasmids include MOBfunctions. The substrates can be incorporated into the same or differentplasmids. Often at least two different types of plasmid having differenttypes of selection marker are used to allow selection for cellscontaining at least two types of vector. Also, where different types ofplasmid are employed, the different plasmids can come from two distinctincompatibility groups to allow stable co-existence of two differentplasmids within the cell. Nevertheless, plasmids from the sameincompatibility group can still co-exist within the same cell forsufficient time to allow homologous recombination to occur.

Plasmids containing diverse substrates are initially introduced intocells by any transfection methods (e.g., chemical transformation,natural competence, transduction, electroporation or biolistics). Often,the plasmids are present at or near saturating concentration (withrespect to maximum transfection capacity) to increase the probability ofmore than one plasmid entering the same cell. The plasmids containingthe various substrates can be transfected simultaneously or in multiplerounds. For example, in the latter approach cells can be transfectedwith a first aliquot of plasmid, transfectants selected and propagated,and then infected with a second aliquot of plasmid.

Having introduced the plasmids into cells, recombination betweensubstrates to generate recombinant genes occurs within cells containingmultiple different plasmids merely by propagating the cells. However,cells that receive only one plasmid are less able to participate inrecombination and the potential contribution of substrates on suchplasmids to evolution is wasted. The rate of evolution can be increasedby allowing all substrates to participate in recombination. Such can beachieved by subjecting transfected cells to electroporation. Theconditions for electroporation are the same as those conventionally usedfor introducing exogenous DNA into cells (e.g., 1,000-2,500 volts, 400μF and a 1-2 mM gap). Under these conditions, plasmids are exchangedbetween cells allowing all substrates to participate in recombination.In addition the products of recombination can undergo further rounds ofrecombination with each other or with the original substrate. The rateof evolution can also be increased by use of conjugative transfer. Toexploit conjugative transfer, substrates can be cloned into plasmidshaving MOB genes and tra genes are also provided in cis or in trans tothe MOB genes. The effect of conjugative transfer is very similar toelectroporation in that it allows plasmids to move between cells andallows recombination between any substrate, and the products of previousrecombination to occur merely by propagating the culture. The details ofhow conjugative transfer is exploited in these vectors are discussed inmore detail below. The rate of evolution can also be increased by use ofmutator host cells (e.g., Mut L, S, D, T, H; human ataxia telengiectasiacells).

The time for which cells are propagated and recombination is allowed tooccur, of course, varies with the cell type but is generally notcritical, because even a small degree of recombination can substantiallyincrease diversity relative to the starting materials. Cells bearingplasmids containing recombined genes are subject to screening orselection for a desired function. For example, if the substrate beingevolved contains a drug resistance gene, one would select for drugresistance. Cells surviving screening or selection can be subjected toone or more rounds of screening/selection followed by recombination orcan be subjected directly to an additional round of recombination.

The next round of recombination can be achieved by several differentformats independently of the previous round. For example, a furtherround of recombination can be effected simply by resuming theelectroporation or conjugation-mediated intercellular transfer ofplasmids described above. Alternatively, a fresh substrate orsubstrates, the same or different from previous substrates, can betransfected into cells surviving selection/screening. Optionally, thenew substrates are included in plasmid vectors bearing a differentselective marker and/or from a different incompatibility group than theoriginal plasmids. As a further alternative, cells survivingselection/screening can be subdivided into two subpopulations, plasmidDNA extracted from one subpopulation and transfected into the other,where the substrates from the plasmids from the two subpopulationsundergo a further round of recombination. In either of the latter twooptions, the rate of evolution can be increased by employingelectroporation, conjugation or mutator cells, as described above. In astill further variation, DNA from cells surviving screening/selectioncan be extracted and subjected to in vitro DNA shuffling.

After the second round of recombination, a second round ofscreening/selection is performed, preferably under conditions ofincreased stringency. If desired, further rounds of recombination andselection/screening can be performed using the same strategy as for thesecond round. With successive rounds of recombination andselection/screening, the surviving recombined substrates evolve towardacquisition of a desired phenotype. Typically, in this and other methodsof in vivo recursive recombination, the final product of recombinationthat has acquired the desired phenotype differs from starting substratesat 0.1%-25% of positions and has evolved at a rate orders of magnitudein excess (e.g., by at least 10-fold, 100-fold, 1000-fold, or 10,000fold) of the rate of naturally acquired mutation of about 1 mutation per10⁻⁹ positions per generation (see Anderson & Hughes, Proc. Natl. Acad.Sci. USA 93, 906-907 (1996)).

FIG. 26 shows an exemplary scheme of plasmid-plasmid recombination.Panel A of the figure shows a library of variant genes cloned into aplasmid. The library is then introduced into cells. Some cells take up asingle plasmid and other cells take up two plasmids as shown in panel B.For cells having taken up two plasmids, the plasmids recombine to givethe products shown in panel C. Plasmids can then be transferred betweencells by electroporation or conjugation as shown in panel D, and furtherrecombination can occur to give the products shown in panel E.Screening/selection then isolates plasmids bearing genes that haveevolved toward acquisition of the property that selection/screening isdesigned to identify. In the course of selection, a cell bearing twoplasmids of which only one contributes to the selected phenotype, maylose the other plasmid, as shown in panel F.

(b) Virus-Plasmid Recombination

The strategy used for plasmid-plasmid recombination can also be used forvirus-plasmid recombination; usually, phage-plasmid recombination.However, some additional comments particular to the use of viruses areappropriate. The initial substrates for recombination are cloned intoboth plasmid and viral vectors. It is usually not critical whichsubstrate(s) are inserted into the viral vector and which into theplasmid, although usually the viral vector should contain differentsubstrate(s) from the plasmid. As before, the plasmid typically containsa selective marker. The plasmid and viral vectors can both be introducedinto cells by transfection as described above. However, a more efficientprocedure is to transfect the cells with plasmid, select transfectantsand infect the transfectants with virus. Because the efficiency ofinfection of many viruses approaches 100% of cells, most cellstransfected and infected by this route contain both a plasmid and virusbearing different substrates.

Homologous recombination occurs between plasmid and virus generatingboth recombined plasmids and recombined virus. For some viruses, such asfilamentous phage, in which intracellular DNA exists in bothdouble-stranded and single-stranded forms, both can participate inrecombination. Provided that the virus is not one that rapidly killscells, recombination can be augmented by use of electroporation orconjugation to transfer plasmids between cells. Recombination can alsobe augmented for some types of virus by allowing the progeny virus fromone cell to reinfect other cells. For some types of virus, virusinfected-cells show resistance to superinfection. However, suchresistance can be overcome by infecting at high multiplicity and/orusing mutant strains of the virus in which resistance to superinfectionis reduced.

The result of infecting plasmid-containing cells with virus depends onthe nature of the virus. Some viruses, such as filamentous phage, stablyexist with a plasmid in the cell and also extrude progeny phage from thecell. Other viruses, such as lambda having a cosmid genome, stably existin a cell like plasmids without producing progeny virions. Otherviruses, such as the T-phage and lytic lambda, undergo recombinationwith the plasmid but ultimately kill the host cell and destroy plasmidDNA. For viruses that infect cells without killing the host, cellscontaining recombinant plasmids and virus can be screened/selected usingthe same approach as for plasmid--plasmid recombination. Progeny virusextruded by cells surviving selection/screening can also be collectedand used as substrates in subsequent rounds of recombination. Forviruses that kill their host cells, recombinant genes resulting fromrecombination reside only in the progeny virus. If the screening orselective assay requires expression of recombinant genes in a cell, therecombinant genes should be transferred from the progeny virus toanother vector, e.g., a plasmid vector, and retransfected into cellsbefore selection/screening is performed.

For filamentous phage, the products of recombination are present in bothcells surviving recombination and in phage extruded from these cells.The dual source of recombinant products provides some additional optionsrelative to the plasmid--plasmid recombination. For example, DNA can beisolated from phage particles for use in a round of in vitrorecombination. Alternatively, the progeny phage can be used to transfector infect cells surviving a previous round of screening/selection, orfresh cells transfected with fresh substrates for recombination. In anaspect, the invention employs recombination between multiplesingle-stranded species, such as single-stranded bacteriophages and/orphagemids.

FIG. 27 illustrates a scheme for virus-plasmid recombination. Panel Ashows a library of variant forms of gene cloned into plasmid and viralvectors. The plasmids are then introduced into cells as shown in panelB. The viral genomes are packaged in vitro and used to infect the cellsin panel B. The viral genomes can undergo replication within the cell,as shown in panel C. The viral genomes undergo recombination withplasmid genomes generating the plasmid and viral forms shown in panel D.Both plasmids and viral genomes can undergo further rounds ofreplication and recombination generating the structures shown in panelsE and F. Screening/selection identifies cells containing plasmid and/orviral genomes having genes that have evolved best to allow survival ofthe cell in the screening/selection process, as shown in panel G. Theseviral genomes are also present in viruses extruded by such cells.

(c) Virus--Virus Recombination

The principles described for plasmid--plasmid and plasmid-viralrecombination can be applied to virus--virus recombination with a fewmodifications. The initial substrates for recombination are cloned intoa viral vector. Usually, the same vector is used for all substrates.Preferably, the virus is one that, naturally or as a result of mutation,does not kill cells. After insertion, viral genomes are usually packagedin vitro. The packaged viruses are used to infect cells at highmultiplicity such that there is a high probability that a cell willreceive multiple viruses bearing different substrates.

After the initial round of infection, subsequent steps depend on thenature of infection as discussed in the previous section.

For example, if the viruses have phagemid genomes such as lambda cosmidsor M13, F1 or Fd phagemids, the phagemids behave as plasmids within thecell and undergo recombination simply by propagating the cells.Recombination is particularly efficient between single-stranded forms ofintracellular DNA. Recombination can be augmented by electroporation ofcells. Following selection/screening, cosmids containing recombinantgenes can be recovered from surviving cells (e.g., by heat induction ofa cos⁻ lysogenic host cell), repackaged in vitro, and used to infectfresh cells at high multiplicity for a further round of recombination.

If the viruses are filamentous phage, recombination of replicating formDNA occurs by propagating the culture of infected cells.Selection/screening identifies colonies of cells containing viralvectors having recombinant genes with improved properties, together withphage extruded from such cells. Subsequent options are essentially thesame as for plasmid-viral recombination.

FIG. 28 shows an example of virus--virus recombination. A library ofdiverse genes is cloned into a lambda cosmid. The recombinant cosmid DNAis packaged in vitro and used to infect host cells at highmultiplicitity such that many cosmids bearing different inserts enterthe same cell. The cell chosen is a cos⁻ lambda lysogen, which oninduction, packages cosmid DNA without packaging lysogenic DNA. Cosmidsrecombine within the cell. Recombination can be accelerated by the useof host cells that are MutD, MutS, MutL and/or express a modified recA.Induction of the lysogen results in release of packaged recombinantcosmids, having greater diversity than the starting materials.

(d) Chromosome-Plasmid Recombination

This format can be used to evolve both the chromosomal and plasmid-bornesubstrates. The format is particularly useful in situations in whichmany chromosomal genes contribute to a phenotype or one does not knowthe exact location of the chromosomal gene(s) to be evolved. The initialsubstrates for recombination are cloned into a plasmid vector. If thechromosomal gene(s) to be evolved are known, the substrates constitute afamily of sequences showing a high degree of sequence identity but somedivergence from the chromosomal gene. If the chromosomal genes to beevolved have not been located, the initial substrates usually constitutea library of DNA segments of which only a small number show sequenceidentity to the gene or gene(s) to be evolved. Divergence betweenplasmid-borne substrate and the chromosomal gene(s) can be induced bymutagenesis or by obtaining the plasmid-borne substrates from adifferent species than that of the cells bearing the chromosome.

The plasmids bearing substrates for recombination are transfected intocells having chromosomal gene(s) to be evolved. Evolution can occursimply by propagating the culture, and can be accelerated bytransferring plasmids between cells by conjugation or electroporation.Evolution can be further accelerated by use of mutator host cells or byseeding a culture of nonmutator host cells being evolved with mutatorhost cells and inducing intercellular transfer of plasmids byelectroporation or conjugation. Preferably, mutator host cells used forseeding contain a negative selection marker to facilitate isolation of apure culture of the nonmutator cells being evolved. Selection/screeningidentifies cells bearing chromosomes and/or plasmids that have evolvedtoward acquisition of a desired function.

Subsequent rounds of recombination and selection/screening proceed insimilar fashion to those described for plasmid--plasmid recombination.For example, further recombination can be effected by propagating cellssurviving recombination in combination with electroporation orconjugative transfer of plasmids. Alternatively, plasmids bearingadditional substrates for recombination can be introduced into thesurviving cells. Preferably, such plasmids are from a differentincompatibility group and bear a different selective marker than theoriginal plasmids to allow selection for cells containing at least twodifferent plasmids. As a further alternative, plasmid and/or chromosomalDNA can be isolated from a subpopulation of surviving cells andtransfected into a second subpopulation. Chromosomal DNA can be clonedinto a plasmid vector before transfection.

FIG. 29 illustrates a scheme for plasmid-chromosome shuffling. Panel Ashows variant forms of a gene cloned into a plasmid vector. The plasmidsare introduced into cells as shown in panel B. In the cells, theplasmids replicate and undergo recombination with a chromosomal copy ofthe gene, as shown in panel C. Exchange of plasmids between cells can beeffected by electroporation or conjugation as shown in panel D. Thechromosomal genes in the two cells shown in panel D have evolved todifferent variant forms. Screening/selection identifies the cell bearingthe chromosomal gene that has evolved that has acquired a desiredproperty that allows the cell to survive screening/selection, as shownin panel E.

(e) Virus-Chromosome Recombination

As in the other methods described above, the virus is usually one thatdoes not kill the cells, and is often a phage or phagemid. The procedureis substantially the same as for plasmid-chromosome recombination.Substrates for recombination are cloned into the vector. Vectorsincluding the substrates can then be transfected into cells or in vitropackaged and introduced into cells by infection. Viral genomes recombinewith host chromosomes merely by propagating a culture. Evolution can beaccelerated by allowing intercellular transfer of viral genomes byelectroporation, or reinfection of cells by progeny virions.Screening/selection identifies cells having chromosomes and/or viralgenomes that have evolved toward acquisition of a desired function.

There are several options for subsequent rounds of recombination. Forexample, viral genomes can be transferred between cells survivingselection/recombination by electroporation. Alternatively, virusesextruded from cells surviving selection/screening can be pooled and usedto superinfect the cells at high multiplicity. Alternatively, freshsubstrates for recombination can be introduced into the cells, either onplasmid or viral vectors.

e. Evolution of Genes by Conjugative Transfer

As noted above, the rate of in vivo evolution of plasmids DNA can beaccelerated by allowing transfer of plasmids between cells byconjugation. Conjugation is the transfer of DNA occurring during contactbetween cells. See Guiney (1993) in: Bacterial Conjugation (Clewell,ed., Plenum Press, New York), pp. 75-104; Reimmann & Haas in BacterialConjugation (Clewell, ed., Plenum Press, New York 1993), at pp.137-188(incorporated by reference in their entirety for all purposes).Conjugation occurs between many types of gram negative bacteria, andsome types of gram positive bacteria. Conjugative transfer is also knownbetween bacteria and plant cells (Agrobacterium tumefaciens) or yeast.

As discussed in copending application attorney docket no. 16528J-014612,the genes responsible for conjugative transfer can themselves be evolvedto expand the range of cell types (e.g., from bacteria to mammals)between which such transfer can occur.

Conjugative transfer is effected by an origin of transfer (oriT) andflanking genes (MOB A, B and C), and 15-25 genes, termed tra, encodingthe structures and enzymes necessary for conjugation to occur. Thetransfer origin is defined as the site required in cis for DNA transfer.Tra genes include tra A, B, C, D, E, F, G, H, I, J, K, L, M, N, P, Q, R,S, T, U, V, W, X, Y, Z, vir AB (alleles 1-11), C, D, E, G, IHF, andFinOP. OriT is sometimes also designated as a tra gene. Other cellularenzymes, including those of the RecBCD pathway, RecA, SSB protein, DNAgyrase, DNA polI, and DNA ligase, are also involved in conjugativetransfer. RecE or recF pathways can substitute for RecBCD.

The tra genes and MOB genes can be expressed in cis or trans to oriT.Vectors undergoing conjugation also have an origin of replication whichis classified as belonging to an incompatibility group such as Inc A, B,C, D, E, F (I-VI), H (I, Y), i (1, 2, 5, ALPHA), J, K, L, M, N, P(ALPHA, BETA, 1 ALPHA, 3, 7, 10, 13) Q, R (H1, H2, H3) S, T, U, W, X, Z.Only vectors from different incompatibility groups can stably co-existin the same cell. However, when two vectors from the sameincompatibility group are transfected into the same cell, the vectorstransiently coexist for sufficient time that recombination can occurbetween the vectors.

One structural protein encoded by a tra gene is the sex pilus, afilament constructed of an aggregate of a single polypeptide protrudingfrom the cell surface. The sex pilus binds to a polysaccharide onrecipient cells and forms a conjugative bridge through which DNA cantransfer. This process activates a site-specific nuclease encoded by aMOB gene, which specifically cleaves DNA to be transferred at oriT. Thecleaved DNA is then threaded through the conjugation bridge by theaction of other tra enzymes.

DNA is transferred more efficiently between cells when present as acomponent of the mobilizable vector. However, some mobilizable vectorsintegrate into the host chromosome and thereby mobilize adjacent genesfrom the chromosome. The F plasmid of E. coli, for example, integratesinto the chromosome at high frequency and mobilizes genes unidirectionalfrom the site of integration. Other mobilizable vectors do notspontaneously integrate into a host chromosome at high efficiency butcan be induced to do by growth under particular conditions (e.g.,treatment with a mutagenic agent, growth at a nonpermissive temperaturefor plasmid replication). See Reimann & Haas in Bacterial Conjugation(ed. Clewell, Plenum Press, NY 1993), Ch. 6.

Conjugation provides a means of recombining gene(s) in vivo to generatediverse recombinant forms of the gene(s). As in other methods ofrecursive recombination, iterative cycles of recombination andselection/screening can be used to evolve the gene(s) toward acquisitionof a new or improved property. As in any method of recursiverecombination, the first step is to generate a library of diverse formsof the gene or genes to be evolved. The diverse forms can be the resultof natural diversity, the application of traditional mutagenesis methods(e.g., error-prone PCR or cassette mutagenesis) or the result of any ofthe other recombination formats discussed in this application, or anycombination of these. The number of diverse forms can vary widely fromabout 10 to 100, 10⁴, 10⁶, 10⁸ or 10¹⁰. Often, the gene(s) of interestare mutagenized as discrete units. However, if the location of gene(s)is not known or a large number of genes are to be evolvedsimultaneously, initial diversity can be generated by in situmutagenesis of a chromosome containing the gene(s).

The library of diverse forms of a gene or gene(s) is introduced intocells containing the apparatus necessary for conjugative transfer(assuming that the library is not already contained in such cells),usually in an arrangement such that the genes can be expressed. Forexample, if the gene(s) are mutagenized in the absence of essentialregulatory sequences such as promoter, these sequences are reattachedbefore introduction into cells. Similarly, if a fragment of a gene ismutagenized in isolation, the mutagenesis products are usuallyreassociated with unchanged flanking sequences before being introducedinto cells. The apparatus necessary for conjugative transfer comprises avector having an origin of transfer together with the mob and tra geneswhose expression is necessary for conjugative transfer to occur. Thesegenes can be included in the vector, in one or more different vectors,or in the chromosome. The library of diverse forms of the gene to beevolved is usually inserted into the vector containing the origin oftransfer (see FIG. 30). However, in some situations the library ofdiverse forms of the gene can be present in the chromosome or a secondvector, as well as, or instead of in the vector containing the origin oftransfer. The library of diverse forms can be inserted in differentplaces in different cells.

A vector bearing a library of variant forms contains at least one originof replication. If transfer between different cell types iscontemplated, the vector can contain two origins of replication, onefunctional in each cell type (i.e., a shuttle vector). Alternatively, ifit is intended that transferred genes should integrate into thechromosome of recipient cells, it is preferable that the vector notcontain an origin of replication functional in the recipient cells(i.e., a suicide vector). The oriT site and/or MOB genes can beintroduced into a vector by cloning or transposing the RK2/RP4 MOBfunction (Guiney, J. Mol. Biol. 162, 699-703 (1982)), or by cointegrateformation with a MOB-containing plasmid. A convenient method for largeplasmids is to use `Tn5-Mob`, which is the Tn5 transposon containing theoriT of RP4. For example, pUC-like mobilizable vectors pK18 and pK19(Schafer et al. (1995) Gene 145:69-73) are suitable starting vectors forcloning the tra gene library to be evolved.

Although not necessary, recombination is sometimes facilitated byinserting the diverse gene library into two different kinds of vectorshaving different incompatibility origins. Each vector should have anoriT and the cell should express MOB and tra functions suitable formobilization of both vectors. Use of two such kinds of vectors allowsstable coexistence of multiple vectors within the same cell andincreases the efficiency of recombination between the vectors.

The collection of cells is propagated in any suitable media to allowgene expression to occur. Tra and mob genes are expressed and mediatetransfer of the mobilizable vector between cells. If the diverse libraryis cloned into a mobilizable vector, its members are transferred ascomponents of the vector. If the diverse library, or certain elements ofthe library, are in trans to the mobilizable vector, they aretransferred only if the mobilizable vector integrates into or proximateto the elements. As discussed above, integration frequently occursspontaneously for the E. coli F plasmid and can be induced for othermobilizable vectors.

As a result of transfer of members of the diverse library between cells,some of the cells come to contain more than one member of the diverselibrary. The multiple members undergo recombination within such cellsgenerating still further diversity in a library of recombinant forms. Ingeneral, the longer cells are propagated the more recombinant forms aregenerated. Generally, recombination results in more than onerecombination product within the same cell. If both recombinationproducts are on vectors and the vectors are from the sameincompatibility group, one of the vectors is lost as the cells arepropagated. This process occurs faster if the cells are propagated onselective media in which one or other of the recombinant productsconfers a selective advantage. After a suitable period of recombination,which depends on the cell type and its growth cycle time, therecombinant forms are subject to screening or selection. Because therecombinant forms are already present in cells, this format forrecombination is particularly amenable to alternation with cycles of invivo screening or selection. The conditions for screening or selection,of course, depend on the property which it is desired that the gene(s)being evolved acquire or improve in. For example, if the property isdrug resistance, recombinant forms having the best drug resistance canbe selected by exposure to the drug. Alternatively, if a cluster ofgenes is being evolved to produce a drug as a secondary metabolite,cells bearing recombinant clusters of the genes can be screened byoverlaying colonies of cell bearing recombinant cluster with a lawn ofcells that are sensitive to the drug. Colonies having recombinantclusters resulting in production of the best drug are identified fromholes in the lawn. If the gene being evolved confers enhanced growthcharacteristics, cells bearing the best genes can be selected by growthcompetition. Antibiotic production can be a growth rate advantage ifcells are competing with other cell types for growth.

Screening/selection produces a subpopulation of cells expressingrecombinant forms of gene(s) that have evolved toward acquisition of adesired property. These recombinant forms can then be subjected tofurther rounds of recombination and screening/selection in any order.For example, a second round of screening/selection can be performedanalogous to the first resulting in greater enrichment for genes havingevolved toward acquisition of the desired property. Optionally, thestringency of selection can be increased between rounds (e.g., ifselecting for drug resistance, the concentration of drug in the mediacan be increased). Further rounds of recombination can also be performedby an analogous strategy to the first round generating furtherrecombinant forms of the gene(s). Alternatively, further rounds ofrecombination can be performed by any of the other molecular breedingformats discussed. Eventually, a recombinant form of the gene(s) isgenerated that has fully acquired the desired property.

FIG. 30 provides an example of how a drug resistance gene can be evolvedby conjugative transfer. Panel A shows a library of diverse genes clonedinto a mobilizable vector bearing as oriT. The vectors are present incells containing a second vector which provides tra functions.Conjugative transfer results in movement of the mobilizable vectorsbetween cells, such that different vectors bearing different variantforms of a gene occupy the same cell, as shown in panel B. The differentforms of the gene recombine to give the products shown in panel C. Afterconjugation and recombination has proceeded for a desired time, cellsare selected to identify those containing the recombined genes, as shownin panel D.

In one aspect, the alternative shuffling method includes the use ofintra-plasmidic recombination, wherein libraries of sequence-recombinedpolynucleotide sequences are obtained by genetic recombination in vivoof direct sequence repeats located on the same plasmid. In a variation,the sequences to be recombined are flanked by site-specificrecombination sequences and the polynucleotides are present in asite-specific recombination system, such as an integron (Hall andCollins (1995) Mol. Microbiol. 15:593, incorporated herein byreference).

In an aspect of the invention, mutator strains of host cells are used toenhance recombination of more highly mismatched sequence-relatedpolynucleotides. Bacterials strains such as MutL, MutS, or MutH or othercells expressing the Mut proteins (XL-1red; Stratagene, San Diego,Calif.) can be used as host cells for shuffling of sequence-relatedpolynucleotides by in vivo recombination. Other mutation-prone host celtypes can also be used, such as those having a proofreading-defectivepolymerase (Foster et al. (1995) Proc. Natl. Acad. Sci. (U.S.A.)92:7951, incorporated herein by reference). Other in vivo mutagenicformats can be employed, including adminstering chemical or radiologicalmutagens to host cells. Examples of such mutagens include but are notlimited to: ENU, MMNG, nitrosourea, BuDR, and the like.

Shuffling can be used to evolve polymerases capable of incorporation ofbase analogs in PCR or PCR-like amplification reactions. A DNApolymerase which is evolved to use base analogs can be used to copy DNAby PCR into a chemical form which gives more resolvable fragmentationpatterns in mass spectrometry, such as for mass spectrometry DNAsequencing. The base analogs can have fewer and/or more favorablefragmentation sites to enhance or facilitate the interpretation of themass spectrum patterns.

Variant polymerases can also be evolved by recursive sequencerecombination to incorporate non-natural nucleotides or nucleotideanalogs, such as phosphorothioate nucleotides. Phosphorothioatenucleotides made with such variant polymerases can provide many uses,including naked DNA gene therapy vectors which are resistant to nucleasedegradation. Other examples of properties of polymerases which can bemodified via recursive sequence recombination include, but are notlimited to, processivity, error rate, proofreading, thermal stability,oxidation resistance, nucleotide preferences, template specificity, andthe like, among others.

In an embodiment, fluorescence-activated cell sorting or analogousmethodology is used to screen for host cells, typically mammalian cells,insect cells, or bacterial cells, comprising a library member of arecursively recombined sequence library, wherein the host cell having alibrary member conferring a desired phenotype can be selected on thebasis of fluorescence or optical density at one or more detectionwavelengths. In one embodiment, for example, each library membertypically encodes an enzyme, which may be secreted from the cell or maybe intracellular, and the enzyme catalyzes conversion of a chromogenicor fluorogenic substrate, which may be capable of diffusing into thehost cell (e.g., if said enzyme is not secreted). Host cells containinglibrary members are contained in fluid drops or gel drops and passed bya detection apparatus where the drops are illuminated with an excitationwavelength and a detector measures either fluorescent emissionwavelength radiation and/or measures optical density (absoption) at oneor more excitatory wavelength(s). The cells suspended in drops arepassed across a sample detector under conditions wherein only about oneindividual cell is present in a sample detection zone at a time. Asource, illuminates each cell and a detector, typically aphotomultiplier or photodiode, detects emitted radiation. The detectorcontrols gating of the cell in the detection zone into one of aplurality of sample collection regions on the basis of the signal(s)detected. A general description of FACS apparatus and methods inprovided in U.S. Pat. Nos. 4,172,227; 4,347,935; 4,661,913; 4,667,830;5,093,234; 5,094,940; and 5,144,224, incorporated herein by reference. Asuitable alternative to convnetional FACS is available from One CellSystems, Inc. Cambridge, Mass.

As can be appreciated from the disclosure above, the present inventionhas a wide variety of applications. Accordingly, the following examplesare offered by way of illustration, not by way of limitation.

EXPERIMENTAL EXAMPLES

In the examples below, the following abbreviations have the followingmeanings. If not defined below, then the abbreviations have their artrecognized meanings.

    ______________________________________                                        ml =       milliliter                                                                     μl = microliters                                                 μM = micromolar                                                            nM = nanomolar                                                                PBS = phosphate buffered saline                                               ng = nanograins                                                               μg = micrograms                                                            IPTG = isopropylthio-β-D-galactoside                                     bp = basepairs                                                                kb = kilobasepairs                                                            dNTP = deoxynucleoside triphosphates                                          PCR = polymerase chain reaction                                               X-gal = 5-bromo-4-chloro-3-indolyl-β-D-galactoside                       DNAseI = deoxyribonuclease                                                    PBS = phosphate buffered saline                                               CDR = complementarity determining regions                                     MIC = minimum inhibitory concentration                                        scFv = single-chain Fv fragment of an antibody                              ______________________________________                                    

In general, standard techniques of recombination DNA technology aredescribed in various publications, e.g. Sambrook et al., 1989, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory; Ausubel etal., 1987, Current Protocols in Molecular Biology, vols. 1 and 2 andsupplements, and Berger and Kimmel, Methods in Enzymology, Volume 152,Guide to Molecular Cloning Techniques (1987), Academic Press, Inc., SanDiego, Calif., each of which is incorporated herein in their entirety byreference. Restriction enzymes and polynucleotide modifying enzymes wereused according to the manufacturers recommendations. Oligonucleotideswere synthesized on an Applied Biosystems Inc. Model 394 DNA synthesizerusing ABI chemicals. If desired, PCR amplimers for amplifying apredetermined DNA sequence may be selected at the discretion of thepractitioner.

EXAMPLES Example 1. LacZ alpha gene reassembly

1) Substrate preparation

The substrate for the reassembly reaction was the dsDNA polymerase chainreaction ("PCR") product of the wild-type LacZ alpha gene from pUC18.(FIG. 2) (28; Gene Bank No. XO2514) The primer sequences were5'AAAGCGTCGATTTTTGTGAT3'(SEQ ID NO:1) and 5'ATGGGGTTCCGCGCACATTT3'(SEQID NO:2). The free primers were removed from the PCR product by WizardPCR prep (Promega, Madison, Wis.) according to the manufacturer'sdirections. The removal of the free primers was found to be important.

2) DNAseI digestion

About 5 μg of the DNA substrate was digested with 0.15 units of DNAseI(Sigma, St. Louis, Mo.) in 100 μl of [50 mM Tris-HCl pH 7.4, 1 mM MgCl₂], for 10-20 minutes at room temperature. The digested DNA was run on a2% low melting point agarose gel. Fragments of 10-70 basepairs (bp) werepurified from the 2% low melting point agarose gels by electrophoresisonto DE81 ion exchange paper (Whatman, Hillsborough, Oreg.). The DNAfragments were eluted from the paper with 1 M NaCl and ethanolprecipitated.

3) DNA Reassembly

The purified fragments were resuspended at a concentration of 10-30ng/μl in PCR Mix (0.2 mM each dNTP, 2.2 mM MgCl₂, 50 mM KCl, 10 mMTris-HCl pH 9.0, 0.1% Triton X-100, 0.3 μl Taq DNA polymerase, 50 μltotal volume). No primers were added at this point. A reassembly programof 94° C. for 60 seconds, 30-45 cycles of [94° C. for 30 seconds, 50-55°C. for 30 seconds, 72° C. for 30 seconds] and 5 minutes at 72° C. wasused in an MJ Research (Watertown, Mass.) PTC-150 thermocycler. The PCRreassembly of small fragments into larger sequences was followed bytaking samples of the reaction after 25, 30, 35 ,40 and 45 cycles ofreassembly (FIG. 2).

Whereas the reassembly of 100-200 bp fragments can yield a single PCRproduct of the correct size, 10-50 base fragments typically yield someproduct of the correct size, as well as products of heterogeneousmolecular weights. Most of this size heterogeneity appears to be due tosingle-stranded sequences at the ends of the products, since afterrestriction enzyme digestion a single band of the correct size isobtained.

4) PCR with primers

After dilution of the reassembly product into the PCR Mix with 0.8 μM ofeach of the above primers (SEQ ID Nos: 1 and 2) and about 15 cycles ofPCR, each cycle consisting of [94° C. for 30 seconds, 50° C. for 30seconds and 72° C. for 30 seconds], a single product of the correct sizewas obtained (FIG. 2).

5) Cloning and analysis

The PCR product from step 4 above was digested with the terminalrestriction enzymes BamHI and EcoO109 and gel purified as describedabove in step 2. The reassembled fragments were ligated into pUC18digested with BamHI and EcoO109. E. Coli were transformed with theligation mixture under standard conditions as recommended by themanufacturer (Stratagene, San Diego, Calif.) and plated on agar plateshaving 100 g/ml ampicillin, 0.004% X-gal and 2 mM IPTG. The resultingcolonies having the HinDIII-NheI fragment which is diagnostic for the ++recombinant were identified because they appeared blue.

This Example illustrates that a 1.0 kb sequence carrying the LacZ alphagene can be digested into 10-70 bp fragments, and that these gelpurified 10-70 bp fragments can be reassembled to a single product ofthe correct size, such that 84% (N=377) of the resulting colonies areLacZ⁺ (versus 94% without shuffling; FIG. 2).

The DNA encoding the LacZ gene from the resulting LacZ⁻ colonies wassequenced with a sequencing kit (United States Biochemical Co.,Cleveland, Ohio) according to the manufacturer's instructions and thegenes were found to have point mutations due to the reassembly process(Table 1). 11/12 types of substitutions were found, and no frameshifts.

                  TABLE 1                                                         ______________________________________                                        Mutations introduced by mutagenic shuffling                                       Transitions                                                                             Frequency   Transversions                                                                          Frequency                                  ______________________________________                                        G - A     6           A - T      1                                              A - G 4 A - C 2                                                               C - T 7 C - A 1                                                               T - C 3 C - G 0                                                                 G - C 3                                                                       G - T 2                                                                       T - A 1                                                                       T - G 2                                                                   ______________________________________                                    

A total of 4,437 bases of shuffled lacZ DNA were sequenced.

The rate of point mutagenesis during DNA reassembly from 10-70 bp pieceswas determined from DNA sequencing to be 0.7% (N=4,473), which issimilar to error-prone PCR. Without being limited to any theory it isbelieved that the rate of point mutagenesis may be lower if largerfragments are used for the reassembly, or if a proofreading polymeraseis added.

When plasmid DNA from 14 of these point-mutated LacZ⁻ colonies werecombined and again reassembled/shuffled by the method described above,34% (N=291) of the resulting colonies were LacZ⁺, and these coloniespresumably arose by recombination of the DNA from different colonies.

The expected rate of reversal of a single point mutation by error-pronePCR, assuming a mutagenesis rate of 0.7% (10), would be expected to be<1%.

Thus large DNA sequences can be reassembled from a random mixture ofsmall fragments by a reaction that is surprisingly efficient and simple.One application of this technique is the recombination or shuffling ofrelated sequences based on homology.

Example 2. LacZ gene and whole plasmid DNA shuffling

1) LacZ gene shuffling

Crossover between two markers separated by 75 bases was measured usingtwo LacZ gene constructs. Stop codons were inserted in two separateareas of the LacZ alpha gene to serve as negative markers. Each markeris a 25 bp non-homologous sequence with four stop codons, of which twoare in the LacZ gene reading frame. The 25 bp non-homologous sequence isindicated in FIG. 3 by a large box. The stop codons are either boxed orunderlined. A 1:1 mixture of the two 1.0 kb LacZ templates containingthe +- and -+ versions of the LacZ alpha gene (FIG. 3) was digested withDNAseI and 100-200 bp fragments were purified as described in Example 1.The shuffling program was conducted under conditions similar to thosedescribed for reassembly in Example 1 except 0.5 μl of polymerase wasadded and the total volume was 100 μl.

After cloning, the number of blue colonies obtained was 24%; (N=386)which is close to the theoretical maximum number of blue colonies (i.e.25%), indicating that recombination between the two markers wascomplete. All of the 10 blue colonies contained the expectedHindIII-NheI restriction fragment.

2) Whole plasmid DNA shuffling

Whole 2.7 kb plasmids (pUC18-+ and pUC18+-) were also tested. A 1:1mixture of the two 2.9 kb plasmids containing the +- and -+ versions ofthe LacZ alpha gene (FIG. 3) was digested with DNAseI and 100-200 bpfragments were purified as described in Example 1. The shuffling programwas conducted under conditions similar to those described for reassemblyin step (1) above except the program was for 60 cycles [94° C. for 30seconds, 55° C. for 30 seconds, 72° C. for 30 seconds]. Gel analysisshowed that after the shuffling program most of the product was greaterthan 20 kb. Thus, whole 2.7 kb plasmids (pUC18-+ and pUC18+-) wereefficiently reassembled from random 100-200 bp fragments without addedprimers.

After digestion with a restriction enzyme having a unique site on theplasmid (EcoO109), most of the product consisted of a single band of theexpected size. This band was gel purified, religated and the DNA used totransform E. coli. The transformants were plated on 0.004% X-gal platesas described in Example 1. 11% (N=328) of the resulting plasmids wereblue and thus ++ recombinants.

3) Spiked DNA Shuffling

Oligonucleotides that are mixed into the shuffling mixture can beincorporated into the final product based on the homology of theflanking sequences of the oligonucleotide to the template DNA (FIG. 4).The LacZ⁻ stop codon mutant (pUC18-+) described above was used as theDNAseI digested template. A 66 mer oligonucleotide, including 18 basesof homology to the wild-type LacZ gene at both ends was added into thereaction at a 4-fold molar excess to correct stop codon mutationspresent in the original gene. The shuffling reaction was conducted underconditions similar to those in step 2 above. The resulting product wasdigested, ligated and inserted into E. coli as described above.

                  TABLE 2                                                         ______________________________________                                                              % blue colonies                                         ______________________________________                                        Control               0.0 (N > 1000)                                            Top strand spike 8.0 (N = 855)                                                Bottom strand spike 9.3 (N = 620)                                             Top and bottom strand spike 2.1 (N = 537)                                   ______________________________________                                    

ssDNA appeared to be more efficient than dsDNA, presumably due tocompetitive hybridization. The degree of incorporation can be variedover a wide range by adjusting the molar excess, annealing temperature,or the length of homology.

Example 3. DNA reassembly in the complete absence of primers

Plasmid pUC18 was digested with restriction enzymes EcoRI, EcoO109, XmnIand AlwNI, yielding fragments of approximately 370, 460, 770 and 1080bp. These fragments were electrophoresed and separately purified from a2% low melting point agarose gel (the 370 and 460 basepair bands couldnot be separated), yielding a large fragment, a medium fragment and amixture of two small fragments in 3 separate tubes.

Each fragment was digested with DNAseI as described in Example 1, andfragments of 50-130 bp were purified from a 2% low melting point agarosegel for each of the original fragments.

PCR mix (as described in Example 1 above) was added to the purifieddigested fragments to a final concentration of 10 ng/μl of fragments. Noprimers were added. A reassembly reaction was performed for 75 cycles[94° C. for 30 seconds, 60° C. for 30 seconds] separately on each of thethree digested DNA fragment mixtures, and the products were analyzed byagarose gel electrophoresis.

The results clearly showed that the 1080, 770 and the 370 and 460 bpbands reformed efficiently from the purified fragments, demonstratingthat shuffling does not require the use of any primers at all.

Example 4. IL-1β gene shuffling

This example illustrates that crossovers based on homologies of lessthan 15 bases may be obtained. As an example, a human and a murine IL-1βgene were shuffled.

A murine IL1-β gene (BBG49) and a human IL1-β gene with E. coli codonusage (BBG2; R&D Systems, Inc., Minneapolis Minn.) were used astemplates in the shuffling reaction. The areas of complete homologybetween the human and the murine IL-1β sequences are on average only 4.1bases long (FIG. 5, regions of heterology are boxed).

Preparation of dsDNA PCR products for each of the genes, removal ofprimers, DNAseI digestion and purification of 10-50 bp fragments wassimilar to that described above in Example 1. The sequences of theprimers used in the PCR reaction were 5'TTAGGCACCCCAGGCTTT3'(SEQ IDNO:3) and 5'ATGTGCTGCAAGGCGATT3' (SEQ ID NO:4).

The first 15 cycles of the shuffling reaction were performed with theKlenow fragment of DNA polymerase I, adding 1 unit of fresh enzyme ateach cycle. The DNA was added to the PCR mix of Example 1 which mixlacked the polymerase. The manual program was 94° C. for 1 minute, andthen 15 cycles of: [95° C. for 1 minute, 10 seconds on dry ice/ethanol(until frozen), incubate about 20 seconds at 25° C., add 1U of Klenowfragment and incubate at 25° C. for 2 minutes]. In each cycle after thedenaturation step, the tube was rapidly cooled in dry ice/ethanol andreheated to the annealing temperature. Then the heat-labile polymerasewas added. The enzyme needs to be added at every cycle. Using thisapproach, a high level of crossovers was obtained, based on only a fewbases of uninterrupted homology (FIG. 5, positions of cross-oversindicated by "₋₋ |⁻ ")

After these 15 manual cycles, Taq polymerase was added and an additional22 cycles of the shuffling reaction [94° C. for 30 seconds, 35° C. for30 seconds] without primers were performed.

The reaction was then diluted 20-fold. The following primers were addedto a final concentration of 0.8 μM:5'AACGCCGCATGCAAGCTTGGATCCTTATT3'(SEQ ID NO:5) and5'AAAGCCCTCTAGATGATTACGAATTCATAT3'(SEQ ID NO:6) and a PCR reaction wasperformed as described above in Example 1. The second primer pairdiffered from the first pair only because a change in restriction siteswas deemed necessary.

After digestion of the PCR product with XbaI and SphI, the fragmentswere ligated into XbaI-SphI-digested pUC18. The sequences of the insertsfrom several colonies were determined by a dideoxy DNA sequencing kit(United States Biochemical Co., Cleveland, Ohio) according to themanufacturer's instructions.

A total of 17 crossovers were found by DNA sequencing of nine colonies.Some of the crossovers were based on only 1-2 bases of uninterruptedhomology.

It was found that to force efficient crossovers based on shorthomologies, a very low effective annealing temperature is required. Withany heat-stable polymerase, the cooling time of the PCR machine (94° C.to 25° C. at 1-2 degrees/second) causes the effective annealingtemperature to be higher than the set annealing temperature. Thus, noneof the protocols based on Taq polymerase yielded crossovers, even when aten-fold excess of one of the IL1-β genes was used. In contrast, aheat-labile polymerase, such as the Klenow fragment of DNA polymerase I,can be used to accurately obtain a low annealing temperature.

Example 5. DNA shuffling of the TEM-1 betalactamase gene

The utility of mutagenic DNA shuffling for directed molecular evolutionwas tested in a betalactamase model system. TEM-1 betalactamase is avery efficient enzyme, limited in its reaction rate primarily bydiffusion. This example determines whether it is possible to change itsreaction specificity and obtain resistance to the drug cefotaxime thatit normally does not hydrolyze.

The minimum inhibitory concentration (MIC) of cefotaxime on bacterialcells lacking a plasmid was determined by plating 10 μl of a 10⁻²dilution of an overnight bacterial culture (about 1000 cfu) of E. coliXL1-blue cells (Stratagene, San Diego, Calif.) on plates with varyinglevels of cefotaxime (Sigma, St. Louis, Mo.), followed by incubation for24 hours at 37° C.

Growth on cefotaxime is sensitive to the density of cells, and thereforesimilar numbers of cells needed to be plated on each plate (obtained byplating on plain LB plates). Platings of 1000 cells were consistentlyperformed.

1) Initial Plasmid Construction

A pUC18 derivative carrying the bacterial TEM-1 betalactamase gene wasused (28). The TEM-1 betalactamase gene confers resistance to bacteriaagainst approximately 0.02 μg/ml of cefotaxime. Sfi1 restriction siteswere added 5' of the promoter and 3' of the end of the gene by PCR ofthe vector sequence with two primers:

    Primer A (SEQ ID NO:7):                                                         5'TTCTATTGACGGCCTGTCAGGCCTCATATATACTTTAGATTGATTT3' and                         - Primer B (SEQ ID NO:8):                                                    5'TTGACGCACTGGCCATGGTGGCCAAAAATAAACAAATAGGGGTTCCGCGCACATTT3'            

and by PCR of the betalactamase gene sequence with two other primers:

    Primer C (SEQ ID NO:9):                                                         5'AACTGACCACGGCCTGACAGGCCGGTCTGACAGTTACCAATGCTT, and                           - Primer D (SEQ ID NO:10):                                                   5'AACCTGTCCTGGCCACCATGGCCTAAATACATTCAAATATGTAT.                         

The two reaction products were digested with SfiI, mixed, ligated andused to transform bacteria.

The resulting plasmid was pUC182Sfi. This plasmid contains an Sfi1fragment carrying the TEM-1 gene and the P-3 promoter.

The minimum inhibitory concentration of cefotaxime for E. coli XL1-blue(Stratagene, San Diego, Calif.) carrying this plasmid was 0.02 μg/mlafter 24 hours at 37° C.

The ability to improve the resistance of the betalactamase gene tocefotaxime without shuffling was determined by stepwise replating of adiluted pool of cells (approximately 10⁷ cfu) on 2-fold increasing druglevels. Resistance up to 1.28 μg/ml could be obtained without shuffling.This represented a 64 fold increase in resistance.

2) DNAseI digestion

The substrate for the first shuffling reaction was dsDNA of 0.9 kbobtained by PCR of pUC182Sfi with primers C and D, both of which containa SfiI site.

The free primers from the PCR product were removed by Wizard PCR prep(Promega, Madison, Wis.) at every cycle.

About 5 μg of the DNA substrate(s) was digested with 0.15 units ofDNAseI (Sigma, St. Louis, Mo.) in 100 Al of 50 mM Tris-HCl pH 7.4, 1 mMMgCl₂, for 10 min at room temperature. Fragments of 100-300 bp werepurified from 2% low melting point agarose gels by electrophoresis ontoDE81 ion exchange paper (Whatman, Hillsborough, Oreg.), elution with 1 MNaCl and ethanol precipitation by the method described in Example 1.

3) Gene shuffling

The purified fragments were resuspended in PCR mix (0.2 mM each dNTP,2.2 mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100), at aconcentration of 10-30 ng/μl. No primers were added at this point. Areassembly program of 94° C. for 60 seconds, then 40 cycles of [94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds] andthen 72° C. for 5 minutes was used in an MJ Research (Watertown, Mass.)PTC-150 thermocycler.

4) Amplification of Reassembly Product with primers

After dilution of the reassembly product into the PCR mix with 0.8 μM ofeach primer (C and D) and 20 PCR cycles [94° C. for 30 seconds, 50° C.for 30 seconds, 72° C. for 30 seconds] a single product 900 bp in sizewas obtained.

5) Cloning and analysis

After digestion of the 900 bp product with the terminal restrictionenzyme SfiI and agarose gel purification, the 900 bp product was ligatedinto the vector pUC182Sfi at the unique SfiI site with T4 DNA ligase(BRL, Gaithersburg Md.). The mixture was electroporated into E. coliXL1-blue cells and plated on LB plates with 0.32-0.64 μg/ml ofcefotaxime (Sigma, St. Louis, Mo.). The cells were grown for up to 24hours at 37° C. and the resulting colonies were scraped off the plate asa pool and used as the PCR template for the next round of shuffling.

6) Subsequent Reassembly Rounds

The transformants obtained after each of three rounds of shuffling wereplated on increasing levels of cefotaxime. The colonies (>100, tomaintain diversity) from the plate with the highest level of cefotaximewere pooled and used as the template for the PCR reaction for the nextround.

A mixture of the cefotaxime^(r) colonies obtained at 0.32-0.64 μg/ml inStep (5) above were used as the template for the next round ofshuffling. 10 ul of cells in LB broth were used as the template in areassembly program of 10 minutes at 99° C., then 35 cycles of [94° C.for 30 seconds, 52° C. for 30 seconds, 72° C. for 30 seconds] and then 5minutes at 72° C. as described above.

The reassembly products were digested and ligated into pUC182Sfi asdescribed in step (5) above. The mixture was electroporated into E. coliXL1-blue cells and plated on LB plates having 5-10 μg/ml of cefotaxime.

Colonies obtained at 5-10 μg/ml were used for a third round similar tothe first and second rounds except the cells were plated on LB plateshaving 80-160 μg/ml of cefotaxime. After the third round, colonies wereobtained at 80-160 μg/ml, and after replating on increasingconcentrations of cefotaxime, colonies could be obtained at up to 320μg/ml after 24 hours at 37° C. (MIC=320 μg/ml).

Growth on cefotaxime is dependent on the cell density, requiring thatall the MICs be standardized (in our case to about 1,000 cells perplate). At higher cell densities, growth at up to 1280 μg/ml wasobtained. The 5 largest colonies grown at 1,280 μg/ml were plated forsingle colonies twice, and the Sfi1 inserts were analyzed by restrictionmapping of the colony PCR products.

One mutant was obtained with a 16,000 fold increased resistance tocefotaxime (MIC=0.02 μg/ml to MIC=320 μg/ml).

After selection, the plasmid of selected clones was transferred backinto wild-type E. coli XL1-blue cells (Stratagene, San Diego, Calif.) toensure that none of the measured drug resistance was due to chromosomalmutations.

Three cycles of shuffling and selection yielded a 1.6×10⁴ -fold increasein the minimum inhibitory concentration of the extended broad spectrumantibiotic cefotaxime for the TEM-1 betalactamase. In contrast, repeatedplating without shuffling resulted in only a 16-fold increase inresistance (error-prone PCR or cassette mutagenesis).

7) Sequence analysis

All 5 of the largest colonies grown at 1,280 μg/ml had a restriction mapidentical to the wild-type TEM-1 enzyme. The SfiI insert of the plasmidobtained from one of these colonies was sequenced by dideoxy DNAsequencing (United States Biochemical Co., Cleveland, Ohio) according tothe manufacturer's instructions. All the base numbers correspond to therevised pBR322 sequence (29), and the amino acid numbers correspond tothe ABL standard numbering scheme (30). The amino acids are designatedby their three letter codes and the nucleotides by their one lettercodes. The term G4205A means that nucleotide 4205 was changed fromguanidine to adenine.

Nine single base substitutions were found. G4205A is located between the-35 and -10 sites of the betalactamase P3 promoter (31) . The promoterup-mutant observed by Chen and Clowes (31) is located outside of theSfi1 fragment used here, and thus could not have been detected. Fourmutations were silent (A3689G, G3713A, G3934A and T3959A), and fourresulted in an amino acid change (C3448T resulting in Gly238Ser, A3615Gresulting in Met182Thr, C3850T resulting in Glu104Lys, and G4107Aresulting in Ala18Val).

8) Molecular Backcross

Molecular backcrossing with an excess of the wild-type DNA was then usedin order to eliminate non-essential mutations.

Molecular backcrossing was conducted on a selected plasmid from thethird round of DNA shuffling by the method identical to normal shufflingas described above, except that the DNAseI digestion and shufflingreaction were performed in the presence of a 40-fold excess of wild-typeTEM-1 gene fragment. To make the backcross more efficient, very smallDNA fragments (30 to 100 bp) were used in the shuffling reaction. Thebackcrossed mutants ere again selected on LB plates with 80-160 μg/ml ofcefotaxime (Sigma, St. Louis, Mo.).

This backcross shuffling was repeated with DNA from colonies from thefirst backcross round in the presence of a 40-fold excess of wild-typeTEM-1 DNA. Small DNA fragments (30-100 bp) were used to increase theefficiency of the backcross. The second round of backcrossed mutantswere again selected on LB plates with 80-160 μg/ml of cefotaxime.

The resulting transformants were plated on 160 μg/ml of cefotaxime, anda pool of colonies was replated on increasing levels of cefotaxime up to1,280 μg/ml. The largest colony obtained at 1,280 μg/ml was replated forsingle colonies.

This backcrossed mutant was 32,000 fold more resistant than wild-type.(MIC=640 μ/ml) The mutant strain is 64-fold more resistant to cefotaximethan previously reported clinical or engineered TEM-1-derived strains.Thus, it appears that DNA shuffling is a fast and powerful tool for atleast several cycles of directed molecular evolution.

The DNA sequence of the SfiI insert of the backcrossed mutant wasdetermined using a dideoxy DNA sequencing kit (United States BiochemicalCo., Cleveland, Ohio) according to the manufacturer's instructions(Table 3). The mutant had 9 single base pair mutations. As expected, allfour of the previously identified silent mutations were lost, revertingto the sequence of the wild-type gene. The promoter mutation (G4205A) aswell as three of the four amino acid mutations (Glu104Lys, Met182Thr,and Gly238Ser) remained in the backcrossed clone, suggesting that theyare essential for high level cefotaxime resistance. However, two newsilent mutations (T3842C and A3767G), as well as three new mutationsresulting in amino acid changes were found (C3441T resulting inArg241His, C3886T resulting in Gly92Ser, and G4035C resulting inAla42Gly). While these two silent mutations do not affect the proteinprimary sequence, they may influence protein expression level (forexample by mRNA structure) and possibly even protein folding (bychanging the codon usage and therefore the pause site, which has beenimplicated in protein folding).

                  TABLE 3                                                         ______________________________________                                        Mutations in Betalactamase                                                         Mutation Type                                                                              Non-Backcrossed                                                                             Backcrossed                                   ______________________________________                                        amino acid    Ala18Lys      --                                                  change Glu104Lys Glu104Lys                                                     Met182Thr Met182Thr                                                           Gly238Ser Gly238Ser                                                           -- Ala42Gly                                                                   -- Gly92Ser                                                                  silent T3959A --                                                               G3934A --                                                                     G3713A --                                                                     A3689G --                                                                     -- T3842C                                                                     -- A3767G                                                                    promoter G4205A G4205A                                                      ______________________________________                                    

Both the backcrossed and the non-backcrossed mutants have a promotermutation (which by itself or in combination results in a 2-3 foldincrease in expression level) as well as three common amino acid changes(Glu104Lys, Met182Thr and Gly238Ser). Glu104Lys and Gly238Ser aremutations that are present in several cefotaxime resistant or otherTEM-1 derivatives (Table 4).

9) Expression Level Comparison

The expression level of the betalactamase gene in the wild-type plasmid,the non-backcrossed mutant and in the backcrossed mutant was compared bySDS-polyacrylamide gel electrophoresis (4-20%; Novex, San Diego, Calif.)of periplasmic extracts prepared by osmotic shock according to themethod of Witholt, B. (32).

Purified TEM-1 betalactamase (Sigma, St. Louis, Mo.) was used as amolecular weight standard, and E. coli XL1-blue cells lacking a plasmidwere used as a negative control.

The mutant and the backcrossed mutant appeared to produce a 2-3 foldhigher level of the betalactamase protein compared to the wild-typegene. The promoter mutation appeared to result in a 2-3 times increasein betalactamase.

Example 6. Construction of mutant combinations of the TEM-1betalactamase gene

To determine the resistance of different combinations of mutations andto compare the new mutants to published mutants, several mutants wereconstructed into an identical plasmid background. Two of the mutations,Glu104Lys and Gly238Ser, are known as cefotaxime mutants. All mutantcombinations constructed had the promoter mutation, to allow comparisonto selected mutants. The results are shown in Table 4.

Specific combinations of mutations were introduced into the wild-typepUC182Sfi by PCR, using two oligonucleotides per mutation.

The oligonucleotides to obtain the following mutations were:

    Ala42Gly                                                                        (SEQ ID NO:11) AGTTGGGTGGACGAGTGGGTTACATCGAACT and                            (SEQ ID NO:12) AACCCACTCGTCCACCCAACTGATCTTCAGCAT;                              - Gln39Lys:                                                                  (SEQ ID NO:13) AGTAAAAGATGCTGAAGATAAGTTGGGTGCAC GAGTGGGTT and                 (SEQ ID NO:14) ACTTATCTTCAGCATCTTTTACTT;                                       - Gly92Ser:                                                                  (SEQ ID NO:15) AAGAGCAACTCAGTCGCCGCATACACTATTCT and                           (SEQ ID NO:16) ATGGCGGCGACTGAGTTGCTCTTGCCCGGCGTCAAT;                           - Glul04Lys:                                                                 (SEQ ID NO:17) TATTCTCAGAATGACTTGGTTAAGTACTCACCAGT CACAGAA and                (SEQ ID NO:18) TTAACCAAGTCATTCTGAGAAT;                                         - Met182Thr:                                                                 (SEQ ID NO:19) AACGACGAGCGTGACACCACGACGCCTGTAGCAATG and                       (SEQ ID NO:20) TCGTGGTGTCACGCTCGTCGTT;                                         - Gly238Ser alone:                                                           (SEQ ID NO:21) TTGCTGATAAATCTGGAGCCAGTGAGCGTGGGTCTC GCGGTA and                (SEQ ID NO:22) TGGCTCCAGATTTATCAGCAA;                                          - Gly238Ser and Arg241His (combined):                                        (SEQ ID NO:23) ATGCTCACTGGCTCCAGATTTATCAGCAAT and                             (SEQ ID NO:24) TCTGGAGCCAGTGAGCATGGGTCTCGCGGTATCATT;                           - G4205A:                                                                    (SEQ ID NO:25) AACCTGTCCTGGCCACCATGGCCTAAATACAATCAAA                          TATGTATCCGCTTATGAGACAATAACCCTGATA.                                      

These separate PCR fragments were gel purified away from the syntheticoligonucleotides. 10 ng of each fragment were combined and a reassemblyreaction was performed at 94° C. for 1 minute and then 25 cycles; [94°C. for 30 sec, 50° C. for 30 seconds and 72° C. for 45 seconds]. PCR wasperformed on the reassembly product for 25 cycles in the presence of theSfiI-containing outside primers (primers C and D from Example 5). TheDNA was digested with Sfi1 and inserted into the wild-type pUC182Sfivector. The following mutant combinations were obtained (Table 4).

                  TABLE 4                                                         ______________________________________                                                                             Source                                     Name           Genotype                       MIC     of MIC                ______________________________________                                        TEM-1 Wild-type              0.02                                                              Glu104Lys                      0.08    10                                     Gly238Ser                      016     10                       - TEM-15         Glu104Lys/Gly238Ser*           10                            - TEM-3          Glu104Lys/Gly238Ser/Gln39Lys   10      37,15                                                              2-32                             - ST-4           Glu104Lys/Gly238Ser/Met182     10                                          Thr*                                                            - ST-1           Glu104Lys/Gly238Ser/Met182     320                                         Thr/Ala18Val/T3959A/G3713A/                                                   G3934A/A3689G*                                                  - ST-2           Glu104Lys/Gly238Ser/Met182Thr/  640                                        Ala42Gly/Gly92Ser/Arg241His/                                                  T3842C/A3767G*                                                  - ST-3           Glu104Lys/Gly238Ser/Met182Thr/  640                                        Ala42Gly/Gly92Ser/Arg241His*                                 ______________________________________                                         *All of these mutants additionally contain the G4205A promoter mutation  

It was concluded that conserved mutations account for 9 of 15 doublingsin the MIC.

Glu104Lys alone was shown to result only in a doubling of the MIC to0.08 μg/ml, and Gly238Ser (in several contexts with one additional aminoacid change) resulted only in a MIC of 0.16 μg/ml (26). The doublemutant Glu104Lys/Gly238Ser has a MIC of 10 μg/ml. This mutantcorresponds to TEM-15.

These same Glu104Lys and Gly238Ser mutations, in combination withGln39Lys (TEM-3) or Thr263Met (TEM-4) result in a high level ofresistance (2-32 μg/ml for TEM-3 and 8-32 μg/ml for TEM-4 (34, 35).

A mutant containing the three amino acid changes that were conservedafter the backcross (Glu104Lys/Met182Thr/Gly238Ser) also had a MIC of 10μg/ml. This meant that the mutations that each of the new selectedmutants had in addition to the three known mutations were responsiblefor a further 32 to 64-fold increase in the resistance of the gene tocefotaxime.

The naturally occurring, clinical TEM-1-derived enzymes (TEM-1-19) eachcontain a different combination of only 5-7 identical mutations(reviews). Since these mutations are in well separated locations in thegene, a mutant with high cefotaxime resistance cannot be obtained bycassette mutagenesis of a single area. This may explain why the maximumMIC that was obtained by the standard cassette mutagenesis approach isonly 0.64 μg/ml (26). For example, both the Glu104Lys as well as theGly238Ser mutations were found separately in this study to have MICsbelow 0.16 μg/ml. Use of DNA shuffling allowed combinatoriality and thusthe Glu104Lys/Gly238Ser combination was found, with a MIC of 10 μg/ml.

An important limitation of this example is the use of a single gene as astarting point. It is contemplated that better combinations can be foundif a large number of related, naturally occurring genes are shuffled.The diversity that is present in such a mixture is more meaningful thanthe random mutations that are generated by mutagenic shuffling. Forexample, it is contemplated that one could use a repertoire of relatedgenes from a single species, such as the pre-existing diversity of theimmune system, or related genes obtained from many different species.

Example 7. Improvement of antibody A10B by DNA shuffling of a library ofall six mutant CDRs.

The A10B scFv antibody, a mouse anti-rabbit IgG, was a gift fromPharmacia (Milwaukee, Wis.). The commercially available Pharmacia phagedisplay system was used, which uses the pCANTAB5 phage display vector.

The original A10B antibody reproducibly had only a low avidity, sinceclones that only bound weakly to immobilized antigen (rabbit IgG), (asmeasured by phage ELISA (Pharmacia assay kit) or by phage titer) wereobtained. The concentration of rabbit IgG which yielded 50% inhibitionof the A10B antibody binding in a competition assay was 13 picomolar.The observed low avidity may also be due to instability of the A10Bclone.

The A10B scFv DNA was sequenced (United States Biochemical Co.,Cleveland, Ohio) according to the manufacturer's instructions. Thesequence was similar to existing antibodies, based on comparison toKabat (33).

1) Preparation of phage DNA

Phage DNA having the A10B wild-type antibody gene (10 ul) was incubatedat 99° C. for 10 min, then at 72° C. for 2 min. PCR mix (50 mM KCl, 10mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 μM each dNTP, 1.9 MM MgCl),0.6 μm of each primer and 0.5 μl Taq DNA Polymerase (Promega, Madison,Wis.) was added to the phage DNA. A PCR program was run for 35 cycles of[30 seconds at 94° C., 30 seconds at 45° C., 45 seconds at 72° C.]. Theprimers used were:

    5'  ATGATTACGCCAAGCTTT 3' (SEQ ID NO:26) and                                     - 5' TTGTCGTCTTTCCAGACGTT 3' (SEQ ID NO:27).                           

The 850 bp PCR product was then electrophoresed and purified from a 2%low melting point agarose gel.

2) Fragmentation

300 ng of the gel purified 850 bp band was digested with 0.18 units ofDNAse I (Sigma, St. Louis, Mo.) in 50 mM Tris-HCl pH 7.5, 10 mM MgCl for20 minutes at room temperature. The digested DNA was separated on a 2%low melting point agarose gel and bands between 50 and 200 bp werepurified from the gel.

3) Construction of Test Library

The purpose of this experiment was to test whether the insertion of theCDRs would be efficient.

The following CDR sequences having internal restriction enzyme siteswere synthesized. "CDR H" means a CDR in the heavy chain and "CDR L"means a CDR in the light chain of the antibody.

    CDR Oligos with restriction sites:                                              CDR H1 (SEQ ID NO:34)                                                         5'TTCTGGCTACATCTTCACAGAATTCATCTAGATTTGGGTGAGGCAGACGCCTGAA3'                    - CDR H2 (SEQ ID NO:35)                                                      5'ACAGGGACTTGAGTGGATTGGAATCACAGTCAAGCTTATCCTTTATCTCAGGTCTCGAGT                TCCAAGTACTTAAAGGGCCACACTGAGTGTA 3'                                             - CDR H3 (SEQ ID NO:36)                                                      5'TGTCTATTTCTGTGCTAGATCTTGACTGCAGTCTTATACGAGGATCCATTGGGGCCAAGG                GACCAGGTCA 3'                                                                  - CDR L1 (SEQ ID NO:37)                                                      5'AGAGGGTCACCATGACCTGCGGACGTCTTTAAGCGATCGGGCTGATGGCCTGGTACCAAC                AGAAGCCTGGAT 3'                                                                - CDR L2 (SEQ ID NO:38)                                                      5'TCCCCCAGACTCCTGATTTATTAAGGGAGATCTAAACAGCTGTTGGTCCCTTTTCGCTTCAGT                                                    3'                                      - CDR L3 (SEQ ID NO:39)                                                      5'ATGCTGCCACTTATTACTGCTTCTGCGCGCTTAAAGGATATCTTCATTTCGGAGGGGGGA                CCAAGCT 3'                                                              

The CDR oligos were added to the purified A10B antibody DNA fragments ofbetween 50 to 200 bp from step (2) above at a 10 fold molar excess. ThePCR mix (50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton x-100, 1.9 mMMgCl, 200 μm each dNTP, 0.3 μl Taq DNA polymerase (Promega, Madison,Wis.), 50 μl total volume) was added and the shuffling program run for 1min at 94° C., 1 min at 72° C., and then 35 cycles: 30 seconds at 94°C., 30 seconds at 55° C., 30 seconds at 72° C.

1 μl of the shuffled mixture was added to 100 μl of a PCR mix (50 mMKCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 μm each dNTP, 1.9 mMMgCl, o.6 μ each of the two outside primers (SEQ ID NO:26 and 27, seebelow), 0.5 μl Taq DNA polymerase) and the PCR program was run for 30cycles of [30 seconds at 94° C., 30 seconds at 45° C., 45 seconds at 72°C.]. The resulting mixture of DNA fragments of 850 basepair size wasphenol/chloroform extracted and ethanol precipitated.

The outside primers were:

    Outside Primer 1: SEQ ID NO:27                                                  5' TTGTCGTCTTTCCAGACGTT 3'                                                     - Outside Primer 2: SEQ ID NO:26                                             5' ATGATTACGCCAAGCTTT 3'                                                

The 850 bp PCR product was digested with the restriction enzymes SfiIand NotI, purified from a low melting point agarose gel, and ligatedinto the pCANTAB5 expression vector obtained from Pharmacia, Milwaukee,Wis. The ligated vector was electroporated according to the method setforth by Invitrogen (San Diego, Calif.) into TG1 cells (Pharmacia,Milwaukee, Wis.) and plated for single colonies.

The DNA from the resulting colonies was added to 100 μl of a PCR mix (50mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 μm each dNTP, 1.9mM MgCl, o.6 μM of Outside primer 1 (SEQ ID No. 27; see below) sixinside primers (SEQ ID NOS:40-45; see below), and 0.5 μl Taq DNApolymerase) and a PCR program was run for 35 cycles of [30 seconds at94° C., 30 seconds at 45° C., 45 seconds at 72° C.]. The sizes of thePCR products were determined by agarose gel electrophoresis, and wereused to determine which CDRs with restriction sites were inserted.

CDR Inside Primers:

    H 1  (SEQ ID NO:40) 5' AGAATTCATCTAGATTTG 3',                                    - H 2  (SEQ ID NO:41) 5' GCTTATCCTTTATCTCAGGTC 3',                            - H 3  (SEQ ID NO:42) 5' ACTGCAGTCTTATACGAGGAT 3'                             - L 1  (SEQ ID NO:43) 5' GACGTCTTTAAGCGATCG 3',                               - L 2  (SEQ ID NO:44) 5' TAAGGGAGATCTAAACAG 3',                               - L 3  (SEQ ID NO:45) 5' TCTGCGCGCTTAAAGGAT 3'                         

The six synthetic CDRs were inserted at the expected locations in thewild-type A10B antibody DNA (FIG. 7). These studies showed that, whileeach of the six CDRs in a specific clone has a small chance of being aCDR with a restriction site, most of the clones carried at least one CDRwith a restriction site, and that any possible combination of CDRs withrestriction sites was generated.

4) Construction of Mutant Complementarity Determining Regions ("CDRs")

Based on our sequence data six oligonucleotides corresponding to the sixCDRs were made. The CDRs (Kabat definition) were syntheticallymutagenized at a ratio of 70 (existing base) :10:10:10, and were flankedon the 5' and 3' sides by about 20 bases of flanking sequence, whichprovide the homology for the incorporation of the CDRs when mixed into amixture of unmutagenized antibody gene fragments in a molar excess. Theresulting mutant sequences are given below.

    Oligos for CDR Library                                                          CDR H1 (SEQ ID NO:28)                                                         5'TTCTGGCTACATCTTCACAACTTATGATATAGACTGGGTGAGGCAGACGCCTGAA 3'                   - CDR H2 (SEQ ID NO:29)                                                      5'ACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGGAGAGGGTGGTACTGAATACAATGA                GAAGTTCAAGGGCAGGGCCACACTGAGTGTA 3'                                             - CDR H3 (SEQ ID NO:30)                                                      5'TGTCTATTTCTGTGCTAGAGGGGACTACTATAGGCGCTACTTTGACTTGTGGGGCCAAGG                GACCACGGTCA 3'                                                                 - CDR L1 (SEQ ID NO:31)                                                      5'AGAGGGTCACCATGACCTGCAGTGCCAGCTCAGGTATACGTTACATATATTGGTACCAAC                AGAAGCCTGGAT 3'                                                                - CDR L2 (SEQ ID NO:32)                                                      5'TCCCCCAGACTCCTGATTTATGACACATCCAACGTGGCTCCTGGAGTCCCTTTTCGCTTCAGT                                                  3'                                        - CDR L3 (SEQ ID N0:33)                                                      5'ATGCTGCCACTTATTACTTGCCAGGAGTGGAGTGGTTATCCGTACACGTTCGGAGGGGGG                ACCAAGCT 3'.                                                                 Bold and underlined sequences were the mutant sequences synthesized using     a mixture of nucleosides of 70:10:10:10 where 70% was the wildtype            nucleoside.                                                              

A 10 fold molar excess of the CDR mutant oligos were added to thepurified A10B antibody DNA fragments between 50 to 200 bp in length fromstep (2) above. The PCR mix (50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1%Triton x-100, 1.9 mM MgCl, 200 μm each dNTP, 0.3 μl Taq DNA polymerase(Promega, Madison, Wis.), 50 μl total volume) was added and theshuffling program run for 1 min at 94° C., 1 min at 72° C., and then 35cycles: [30 seconds at 94° C., 30 seconds at 55° C., 30 seconds at 72°C.].

1 μl of the shuffled mixture was added to 100 μl of a PCR mix (50 mMKCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 200 μm each dNTP, 1.9 mMMgCl, o.6 μM each of the two outside primers (SEQ ID NO:26 and 27, seebelow), 0.5 μl Taq DNA polymerase) and the PCR program was run for 30cycles of [30 seconds at 94° C., 30 seconds at 45° C., 45 seconds at 72°C.]. The resulting mixture of DNA fragments of 850 basepair size wasphenol/chloroform extracted and ethanol precipitated.

The outside primers were:

    Outside Primer 1: SEQ ID NO:27 5' TTGTCGTCTTTCCAGACGTT 3'                        - Outside Primer 2: SEQ ID NO:26 5' ATGATTACGCCAAGCTTT 3'              

5) Cloning of the scFv antibody DNA into pCANTAB5

The 850 bp PCR product was digested with the restriction enzymes SfiIand NotI, purified from a low melting point agarose gel, and ligatedinto the pCANTAB5 expression vector obtained from Pharmacia, Milwaukee,Wis. The ligated vector was electroporated according to the method setforth by Invitrogen (San Diego, Calif.) into TG1 cells (Pharmacia,Milwaukee, Wis.) and the phage library was grown up using helper phagefollowing the guidelines recommended by the manufacturer.

The library that was generated in this fashion was screened for thepresence of improved antibodies, using six cycles of selection.

6) Selection of high affinity clones

15 wells of a 96 well microtiter plate were coated with Rabbit IgG(Jackson Immunoresearch, Bar Harbor Me.) at 10 μg /well for 1 hour at37° C., and then blocked with 2% non-fat dry milk in PBS for 1 hour at37° C.

100 μl of the phage library (1×10¹⁰ cfu) was blocked with 100 μl of 2%milk for 30 minutes at room temperature, and then added to each of the15 wells and incubated for 1 hour at 37° C.

Then the wells were washed three times with PBS containing 0.5% Tween-20at 37° C. for 10 minutes per wash. Bound phage was eluted with 100 μlelution buffer (Glycine-HCl, pH 2.2), followed by immediateneutralization with 2M Tris pH 7.4 and transfection for phageproduction. This selection cycle was repeated six times.

After the sixth cycle, individual phage clones were picked and therelative affinities were compared by phage ELISA, and the specificityfor the rabbit IgG was assayed with a kit from Pharmacia (Milwaukee,Wis.) according to the methods recommended by the manufacturer.

The best clone has an approximately 100-fold improved expression levelcompared with the wild-type A10B when tested by the Western assay. Theconcentration of the rabbit IgG which yielded 50% inhibition in acompetition assay with the best clone was 1 picomolar. The best clonewas reproducibly specific for rabbit antigen. The number of copies ofthe antibody displayed by the phage appears to be increased.

Example 8. In vivo recombination via direct repeats of partial genes

A plasmid was constructed with two partial, inactive copies of the samegene (beta-lactamase) to demonstrate that recombination between thecommon areas of these two direct repeats leads to full-length, activerecombinant genes.

A pUC18 derivative carrying the bacterial TEM-1 betalactamase gene wasused (Yanish-Perron et al., 1985, Gene 33:103-119). The TEM-1betalactamase gene ("Bla") confers resistance to bacteria againstapproximately 0.02 μg/ml of cefotaxime. Sfi1 restriction sites wereadded 5' of the promoter and 3' of the end of the betalactamase gene byPCR of the vector sequence with two primers:

    Primer A (SEQ ID NO: 46)                                                        5' TTCTATTGACGGCCTGTCAGGCCTCATATATACTTTAGATTGATTT 3'                           - PRIMER B (SEQ ID NO: 47)                                                   5' TTGACGCACTGGCCATGGTGGCCAAAAATAAACAAATAGGGGTTCCGCGCAC                       ATTT 3'                                                                 

and by PCR of the beta-lactamase gene sequence with two other primers:

    Primer C (SEQ ID NO: 48)                                                        5' AACTGACCACGGCCTGACAGGCCGGTCTGACAGTTACCAATGCTT 3'                            - Primer D (SEQ ID NO: 49)                                                   5' AACCTGTCCTGGCCACCATGGCCTAAATACATTCAAATATGTAT 3'                      

The two reaction products were digested with Sfi1, mixed, ligated andused to transform competent E. coli bacteria by the procedure describedbelow. The resulting plasmid was pUC182Sfi-Bla-Sfi. This plasmidcontains an Sfi1 fragment carrying the Bla gene and the P-3 promoter.

The minimum inhibitory concentration of cefotaxime for E. coli XL1-blue(Stratagene, San Diego, Calif.) carrying pUC182Sfi-Bla-Sfi was 0.02μg/ml after 24 hours at 37° C.

The tetracycline gene of pBR322 was cloned into pUC18Sfi-Bla-Sfi usingthe homologous areas, resulting in pBR322TetSfi-Bla-Sfi. The TEM-1 genewas then deleted by restriction digestion of the pBR322TetSfi-Bla-Sfiwith SspI and FspI and blunt-end ligation, resulting inpUC322TetSfi-Sfi.

Overlapping regions of the TEM-1 gene were amplified using standard PCRtechniques and the following primers:

    Primer 2650 (SEQ ID NO: 50) 5' TTCTTAGACGTCAGGTGGCACTT 3'                        - Primer 2493 (SEQ ID NO: 51) 5' TTT TAA ATC AAT CTA AAG TAT 3'                                                    - Primer 2651 (SEQ ID NO: 52) 5'                                             TGCTCATCCACGAGTGTGGAGAAGTGGTCCTGCAA                                          CTTTAT 3, 'and                             - Primer 2652 (SEQ ID NO: 53)                                                ACCACTTCTCCACACTCGTGGATGAGCACTTTTAAAGTT                                 

The two resulting DNA fragments were digested with Sfi1 and BstX1 andligated into the Sfi site of pBR322TetSfi-Sfi. The resulting plasmid wascalled pBR322Sfi-BL-LA-Sfi. A map of the plasmid as well as a schematicof intraplasmidic recombination and reconstitution of functionalbeta-lactamase is shown in FIG. 9.

The plasmid was electroporated into either TG-1 or JC8679 E. coli cells.E. coli JC8679 is RecBC sbcA (Oliner et al., 1993, NAR 21:5192). Thecells were plated on solid agar plates containing tetracycline. Thosecolonies which grew, were then plated on solid agar plates containing100 μg/ml ampicillin and the number of viable colonies counted. Thebeta-lactamase gene inserts in those transformants which exhibitedampicillin resistance were amplified by standard PCR techniques using

    Primer 2650 (SEQ ID NO: 50) 5' TTCTTAGACGTCAGGTGGCACTT 3' and                    - Primer 2493 (SEQ ID NO: 51) 5' TTTTAAATCAATCTAAAGTAT 3' and the      

length of the insert measured. The presence of a 1 kb insert indicatesthat the gene was successfully recombined, as shown in FIG. 9 and Table5.

                  TABLE 5                                                         ______________________________________                                        Cell      Tet Colonies                                                                            Amp colonies  Colony PCR                                  ______________________________________                                        TG-1      131       21            3/3 at 1 kb                                   JC8679 123 31 4/4 at 1 kb                                                     vector 51 0                                                                   control                                                                     ______________________________________                                    

About 17-25% of the tetracycline-resistant colonies were alsoampicillin-resistant and all of the Ampicillin resistant colonies hadcorrectly recombined, as determined by colony PCR. Therefore, partialgenes located on the same plasmid will successfully recombine to createa functional gene.

Example 9. In vivo recombination via direct repeats of full-lengthgenes.

A plasmid with two full-length copies of different alleles of thebeta-lactamase gene was constructed. Homologous recombination of the twogenes resulted in a single recombinant full-length copy of that gene.

The construction of pBR322TetSfi-Sfi and pBR322TetSfi-Bla-Sfi wasdescribed above.

The two alleles of the beta-lactamase gene were constructed as follows.Two PCR reactions were conducted with pUC18Sfi-Bla-Sfi as the template.One reaction was conducted with the following primers.

    Primer 2650 (SEQ ID NO: 50) 5' TTCTTAGACGTCAGGTGGCACTT 3'                        - Primer 2649 (SEQ ID NO: 51)                                                5'   ATGGTAGTCCACGAGTGTGGTAGTGACAGGCCGGTCTGACAGTTA                            CCAATGCTT 3'                                                            

The second PCR reaction was conducted with the following primers:

    Primer 2648 (SEQ ID NO: 54)                                                     5' TGTCACTACCACACTCGTGGACTACCATGGCCTAAATACATTCAAA                             TATGTAT 3'                                                                     - Primer 2493 (SEQ ID NO: 51) 5' TTT TAA ATC AAT CTA AAG TAT 3'        

This yielded two Bla genes, one with a 5' Sfi1 site and a 3' BstX1 site,the other with a 5' BstX1 site and a 3' Sfi1 site.

After digestion of these two genes with BstX1 and Sfi1, and ligationinto the Sfi1-digested plasmid pBR322TetSfi-Sfi, a plasmid(pBR322-Sfi-2BLA-Sfi) with a tandem repeat of the Bla gene was obtained.(See FIG. 10).

The plasmid was electroporated into E. coli cells. The cells were platedon solid agar plates containing 15 μg/ml tetracycline. Those colonieswhich grew, were then plated on solid agar plates containing 100 μg/mlampicillin and the number of viable colonies counted. The Bla inserts inthose transformants which exhibited ampicillin resistance were amplifiedby standard PCR techniques using the method and primers described inExample 8. The presence of a 1 kb insert indicated that the duplicategenes had recombined, as indicated in Table 6.

                  TABLE 6                                                         ______________________________________                                        Cell      Tet Colonies                                                                            Amp colonies  Colony PCR                                  ______________________________________                                        TG-1      28        54            7/7 at 1 kb                                   JC8679 149 117 3/3 at 1 kb                                                    vector 51 0                                                                   control                                                                     ______________________________________                                    

Colony PCR confirmed that the tandem repeat was efficiently recombinedto form a single recombinant gene.

Example 10. Multiple cycles of direct repeat recombination -Interplasmidic

In order to determine whether multiple cycles of recombination could beused to produce resistant cells more quickly, multiple cycles of themethod described in Example 9 were performed.

The minus recombination control consisted of a single copy of thebetalactamase gene, whereas the plus recombination experiment consistedof inserting two copies of betalactamase as a direct repeat. Thetetracycline marker was used to equalize the number of colonies thatwere selected for cefotaxime resistance in each round, to compensate forligation efficiencies.

In the first round, pBR322TetSfi-Bla-Sfi was digested with EcrI andsubject to PCR with a 1:1 mix (1 ml) of normal and Cadwell PCR mix(Cadwell and Joyce (1992) PCR Methods and Applications 2:28-33) forerror prone PCR. The PCR program was 70° C. for 2 minutes initially andthen 30 cycles of 94° C. for 30 seconds, 52° C. for 30 second and 72° C.for 3 minutes and 6 seconds per cycle, followed by 72° C. for 10minutes.

The primers used in the PCR reaction to create the one Bla gene controlplasmid were Primer 2650 (SEQ ID NO: 50) and Primer 2719 (SEQ ID NO: 55)5' TTAAGGGATTTTGGTCATGAGATT 3'. This resulted in a mixed population ofamplified DNA fragments, designated collectively as Fragment #59. Thesefragments had a number of different mutations.

The primers used in two different PCR reactions to create the two Blagene plasmids were Primer 2650 (SEQ ID NO: 50) and Primer 2649 (SEQ IDNO: 51) for the first gene and Primers 2648 (SEQ ID NO: 54) and Primer2719 (SEQ ID NO: 55) for the second gene. This resulted in a mixedpopulation of each of the two amplified DNA fragments: Fragment #89(amplified with primers 2648 and 2719) and Fragment #90 (amplified withprimers 2650 and 2649). In each case a number of different mutations hadbeen introduced the mixed population of each of the fragments.

After error prone PCR, the population of amplified DNA fragment #59 wasdigested with Sfi1, and then cloned into pBR322TetSfi-Sfi to create amixed population of the plasmid pBR322Sfi-Bla-Sfi¹.

After error prone PCR, the population of amplified DNA fragments #90 and#89 was digested with SfiI and BstXI at 50° C., and ligated intopBR322TetSfi-Sfi to create a mixed population of the plasmidpBR322TetSfi-2Bla-Sfi¹ (FIG. 10).

The plasmids pBR322Sfi-Bla-Sfi¹ and pBR322Sfi-2Bla-Sfi¹ wereelectroporated into E. coli JC8679 and placed on agar plates havingdiffering concentrations of cefotaxime to select for resistant strainsand on tetracycline plates to titre.

An equal number of colonies (based on the number of colonies growing ontetracycline) were picked, grown in LB-tet and DNA extracted from thecolonies. This was one round of the recombination. This DNA was digestedwith EcrI and used for a second round of error-prone PCR as describedabove.

After five rounds the MIC (minimum inhibitory concentration) forcefotaxime for the one fragment plasmid was 0.32 whereas the MIC for thetwo fragment plasmid was 1.28. The results show that after five cyclesthe resistance obtained with recombination was four-fold higher in thepresence of in vivo recombination.

Example 11. In vivo recombination via electroporation of fragments

Competent E. coli cells containing pUC18Sfi-Bla-Sfi were prepared asdescribed. Plasmid pUC18Sfi-Bla-Sfi contains the standard TEM-1beta-lactamase gene as described, supra.

A TEM-1 derived cefotaxime resistance gene from pUC18fi-cef-Sfi, (cloneST2) (Stemmer WPC (1994) Nature 370:389-91, incorporated herein byreference) which confers on E. coli carrying the plasmid an MIC of 640μg/ml for cefotaxime, was obtained. In one experiment the completeplasmid pUC18Sfi-cef-Sfi DNA was electroporated into E. coli cellshaving the plasmid pUC18Sfi-Bla-Sfi.

In another experiment the DNA fragment containing the cefotaxime genefrom pUC18Sfi-cef-Sfi was amplified by PCR using the primers 2650 (SEQID NO: 50) and 2719 (SEQ ID NO: 55). The resulting 1 kb PCR product wasdigested into DNA fragments of <100 bp by DNase and these fragments wereelectroporated into the competent E. coli cells which already containedpUC18Sfi-Bla-Sfi.

The transformed cells from both experiments were then assayed for theirresistance to cefotaxime by plating the transformed cells onto agarplates having varying concentrations of cefotaxime. The results areindicated in Table 7.

                  TABLE 7                                                         ______________________________________                                        Colonies/ Cefotaxime Concentration                                                              0.16    0.32  1.28  5.0   10.0                              ______________________________________                                        no DNA control                                                                              14                                                                ST-2 mutant, whole  4000 2000 800 400                                         ST-2 mutant, fragments  1000 120 22 7                                         Wildtype, whole 27                                                            Wildtype, fragments 18                                                      ______________________________________                                    

From the results it appears that the whole ST-2 Cef gene was insertedinto either the bacterial genome or the plasmid after electroporation.Because most insertions are homologous, it is expected that the gene wasinserted into the plasmid, replacing the wildtype gene. The fragments ofthe Cef gene from St-2 also inserted efficiently into the wild-type genein the plasmid. No sharp increase in cefotaxime resistance was observedwith the introduction of the wildtype gene (whole or in fragments) andno DNA. Therefore, the ST-2 fragments were shown to yield much greatercefotaxime resistance than the wild-type fragments. It was contemplatedthat repeated insertions of fragments, prepared from increasingresistant gene pools would lead to increasing resistance.

Accordingly, those colonies that produced increased cefotaximeresistance with the St-2 gene fragments were isolated and the plasmidDNA extracted. This DNA was amplified using PCR by the method describedabove. The amplified DNA was digested with DNase into fragments (<100bp) and 2-4 μg of the fragments were electroporated into competent E.coli cells already containing pUC322Sfi-Bla-Sfi as described above. Thetransformed cells were plated on agar containing varying concentrationsof cefotaxime.

As a control, competent E. coli cells having the plasmidpUC18Sfi-Kan-Sfi were also used. DNA fragments from the digestion of thePCR product of pUC18Sfi-cef-Sfi were electroporated into these cells.There is no homology between the kanamycin gene and the beta-lactamasegene and thus recombination should not occur.

This experiment was repeated for 2 rounds and the results are shown inTable 8.

                  TABLE 8                                                         ______________________________________                                                                         Cef resistant                                  Round Cef conc. KAN control colonies                                        ______________________________________                                        1         0.16-0.64  lawn        lawn                                           replate 0.32 10 small 1000                                                    2 10 10 400                                                                   Replate  100 sm @ 2.5 50 @ 10                                                 3 40 100 sm                                                                    1280  100 sm                                                               ______________________________________                                    

Example 12 Determination of Recombination Formats

This experiment was designed to determine which format of recombinationgenerated the most recombinants per cycle.

In the first approach, the vector pUC18Sfi-Bla-Sfi was amplified withPCR primers to generate a large and small fragment. The large fragmenthad the plasmid and ends having portions of the Bla gene, and the smallfragment coded for the middle of the Bla gene. A third fragment havingthe complete Bla gene was created using PCR by the method in Example 8.The larger plasmid fragment and the fragment containing the complete Blagene were electroporated into E. coli JC8679 cells at the same time bythe method described above and the transformants plated on differingconcentrations of cefotaxime.

In approach 2, the vector pUC18Sfi-Bla-Sfi was amplified to produce thelarge plasmid fragment isolated as in approach 1 above. The twofragments each comprising a portion of the complete Bla gene, such thatthe two fragments together spanned the complete Bla gene werealsoobtained by PCR. The large plasmid fragment and the two Bla genefragments were all electroporated into competent E. coli JC8679 cellsand the transformants plated on varying concentrations of cefotaxime.

In the third approach, both the vector and the plasmid wereelectroporated into E. coli JC8679 cells and the transformants wereplated on varying concentrations of cefotaxime.

In the fourth approach, the complete Bla gene was electroporated into E.coli JC8679 cells already containing the vector pUCSfi-Sfi and thetransformants were plated on varying concentrations of cefotaxime. Ascontrols, the E. coli JC8679 cells were electroporated with either thecomplete Bla gene or the vector alone.

The results are presented in FIG. 11. The efficiency of the insertion oftwo fragments into the vector is 100× lower than when one fragmenthaving the complete Bla gene is used. Approach 3 indicated that theefficiency of insertion does depend on the presence of free DNA endssince no recombinants were obtained with this approach. However, theresults of approach 3 were also due to the low efficiency ofelectroporation of the vector. When the expression vector is already inthe competent cells, the efficiency of the vector electroporation is notlonger a factor and efficient homologous recombination can be achievedeven with uncut vector.

Example 12. Kit for cassette shuffling to optimize vector performance

In order to provide a vector capable of conferring an optimizedphenotype (e.g., maximal expression of a vector-encoded sequence, suchas a cloned gene), a kit is provided comprising a variety of cassetteswhich can be shuffled, and optimized shufflants can be selected. FIG. 12shows schematically one embodiment, with each loci having a plurality ofcassettes. For example, in a bacterial expression system, FIG. 13 showsexample cassettes that are used at the respective loci. Each cassette ofa given locus (e.g., all promoters in this example) are flanked bysubstantially identical sequences capable of overlapping the flankingsequence(s) of cassettes of an adjacent locus and preferably alsocapable of participating in homologous recombination or non-homologousrecombination (e.g., lox/cre or flp/frt systems), so as to affordshuffling of cassettes within a locus but substantially not betweenloci.

Cassettes are supplied in the kit as PCR fragments, which each cassettetype or individual cassette species packaged in a separate tube. Vectorlibraries are created by combining the contents of tubes to assemblewhole plasmids or substantial portions thereof by hybridization of theoverlapping flanking sequences of cassettes at each locus with cassettesat the adjacent loci. The assembled vector is ligated to a predeterminedgene of interest to form a vector library wherein each library membercomprises the predetermined gene of interest and a combination ofcassettes determined by the association of cassettes. The vectors aretransferred into a suitable host cell and the cells are cultured underconditions suitable for expression, and the desired phenotype isselected.

Example 13. Shuffling to optimize Green Fluorescent Protein (GFP)properties

Background

Green fluorescent protein ("GFP") is a polypeptide derived from anapopeptide having 238 amino acid residues and a molecular weight ofapproximately 27,000. GFP contains a chromophore formed from amino acidresidues 65 through 67. As its name indicates, GFP fluoresces; it doesnot bioluminesce like luciferase. In vivo, the chromophore of GFP isactivated by energy transfer from coelenterazine complexed with thephotoprotein aequorin, with GFP exhibiting green fluorescence at 510 nm.Upon irradiation with blue or UV light, GFP exhibits green fluorescenceat approximately 510 nm.

The green fluorescent protein (GFP) of the jellyfish Aequorea victoriais a very useful reporter for gene expression and regulation (Prasher etal. (1992) Gene 111:229; Prasher et al. (1995) Trends In Genetics11:320; Chalfie et al. (1994) Science 263:802, incorporated herein byreference). WO95/21191 discloses a polynucleotide sequence encoding a238 amino acid GFP apoprotein which contains a chromophore formed fromamino acids 65 through 67. WO95/21191 disclose that a modification ofthe cDNA for the apopeptide of A. victoria GFP results in synthesis of apeptide having altered fluorescent properties. A mutant GFP (S65T)resulting in a 4-6-fold improvement in excitation amplitude has beenreported (Heim et al. (1994) Proc. Natl. Acad. Sci. (U.S.A.) 91:12501).

Overview

Green fluorescent protein (GFP) has rapidly become a widely usedreporter of gene regulation. However, in many organisms, particularlyeukaryotes, the whole cell fluorescence signal was found to be too low.The goal was to improve the whole cell fluorescence of GFP for use as areporter for gene regulation for E. coli and mammalian cells. Theimprovement of GFP by rational design appeared difficult because thequantum yield of GFP is already 0.7-0.8 (Ward et al. (1982) Photochem.Photobiol. 35:803) and the expression level of GFP in a standard E. coliconstruct was already about 75% of total protein.

Improvement of GFP was performed first by synthesis of a GFP gene withimproved codon usage. The GFP gene was then further improved by thedisclosed method(s), consisting of recursive cycles of DNA shuffling orsexual PCR of the GFP gene, combined with visual selection of thebrightest clones. The whole cell fluorescence signal in E. coli wasoptimized and selected mutants were then assayed to determineperformance of the best GFP mutants in eukaryotic ceils.

A synthetic gene was synthesized having improved codon usage and havinga 2.8-fold improvement of the E. coli whole cell fluorescence signalcompared to the industry standard GFP construct (Clontech, Palo Alto,Calif.). An additional 16-fold improvement was obtained from threecycles of sexual PCR and visual screening for the brightest E. colicolonies, for a 45-fold improvement over the standard construct.Expressed in Chinese Hamster Ovary (CHO) cells, this shuffled mutantshowed a 42-fold improvement of signal over the synthetic construct. Theexpression level in E. coli was unaltered at about 75% of total protein.The emission and excitation maxima of the GFP were also unchanged.Whereas in E. coli most of the wildtype GFP ends up in inclusion bodies,unable to activate its chromophore, most of the mutant protein(s) weresoluble and active. The three amino acid mutations thus guide the mutantprotein into the native folding pathway rather than toward aggregation.The results show that DNA sequence shuffling (sexual PCR) can solvecomplex practical problems and generate advantageous mutant variantsrapidly and efficiently.

MATERIALS AND METHODS

GFP gene construction

A gene encoding the GFP protein with the published sequence (Prasher etal. (1995) op.cit, incorporated herein by reference) (238 AA, 27 kD) wasconstructed from oligonucleotides. In contrast to the commerciallyavailable GFP construct (Clontech, Palo Alto, Calif.), the sequenceincluded the Ala residue after the fMet, as found in the original cDNAclone. Fourteen oligonucleotides ranging from 54 to 85 bases wereassembled as seven pairs by PCR extension. These segments were digestedwith restriction enzymes and cloned separately into the vector Alpha+GFP(Whitehorn et al. (1995) Bio/Technology 13:1215, incorporated herein byreference) and sequenced. These segments were then ligated into theeukaryotic expression vector Alpha+to form the full-length GFPconstruct, Alpha+GFP (FIG. 14). The resulting GFP gene contained alteredArginine codons at amino acid positions 73 (CGT), 80 (CGG), 96 (CGC) and122 (CGT). To reduce codon bias and facilitate expression in E. coli, anumber of other silent mutations were engineered into the sequence tocreate the restriction sites used in the assembly of the gene. Thesewere S2 (AGT to AGC; to create an NheI site), K41 (AAA to AAG; HinDIII),Y74 (TAC to TAT) and P75 (CCA to CCG; BspE1), T108 (AGA to AGG; NnuI),L141 (CTC to TTG) and E142 (GAA to GAG; XhoI), S175 (TCC to AGC; BamHI)and S202 (TCG to TCC; SalI). The 5' and 3' untranslated ends of the genecontained XbaI and EcoRI sites, respectively. The sequence of the genewas confirmed by sequencing.

Other suitable GFP vectors and sequences can be obtained from theGenBank database, such as via Internet World Wide Web, as files:CVU36202, CVU36201, XXP35SGFP, XXU19282, XXU19279, XXU19277, XXU19276,AVGFP2, AVGFP1, XXU19281, XXU19280, XXU19278, AEVGFP, and XXU17997,which are incorporated herein by reference to the same extent as if thesequence files and comments were printed and inserted herein.

The XbaI-EcoRI fragment of Alpha+GFP, containing the whole GFP gene, wassubcloned into the prokaryotic expression vector pBAD18 (Guzman et al.(1995) J. Bacteriol. 177:4121), resulting in the bacterial expressionvector pBAD18-GFP (FIG. 14). In this vector GFP gene expression is underthe control of the arabinose promoter/repressor (araBAD), which isinducible with arabinose (0.2%). Because this is the only construct withthe original amino acid sequence, it is referred to as wildtype GFP(`wt`). A GFP-expressing bacterial vector was obtained from Clontech(Palo Alto, Calif.), which is referred to herein as `Clontech`construct. GFP expression from the `Clontech` construct requires IPTGinduction.

Gene shuffling and selection

An approximately 1 kb DNA fragment containing the whole GFP gene wasobtained from the PBAD-GFP vector by PCR with primers5'-TAGCGGATCCTACCTGACGC (near NheI site) and 5'GAAAATCTTCTCTCATCCG (nearEcoRI site) and purified by Wizard PCR prep (Promega, Madison, Wis.).This PCR product was digested into random fragments with DNase I (Sigma)and 50-300 bp fragments were purified from 2% low melting point agarosegels. The purified fragments were resuspended at 10-30 ng/ul in PCRmixture (Promega, Madison, Wis.; 0.2 mM each dNTP/2.2 mM MgCl₂ /50 MMKCl/10 mM Tris-HCl, pH 9.0/0.1% Triton-X-100) with Taq DNA polymerase(Promega) and assembled (without primers) using a PCR program of 35cycles of 94° C. 30s, 45° C. 30s, 72° C. 30s, as described in Stemmer,WPC (1994) Nature 370:389, incorporated herein by reference. The productof this reaction was diluted 40× into new PCR mix, and the full lengthproduct was amplified with the same two primers in a PCR of 25 cycles of94° C. 30s, 50° C. 30s, 72° C. 30s, followed by 72° C. for 10 min. Afterdigestion of the reassembled product with NheI and EcoRI, this libraryof point-mutated and in vitro recombined GFP genes was cloned back intothe PBAD vector, electroporated into E. coli TG1 (Pharmacia), and platedon LB plates with 100 ug/ml ampicillin and 0.2 % arabinose to induce GFPexpression from the arabinose promoter.

Mutant selection

Over a standard UV light box (365 nm) the 40 brightest colonies wereselected and pooled. The pool of colonies was used as the template for aPCR reaction to obtain a pool of GFP genes. Cycles 2 and 3 wereperformed identical to cycle 1. The best mutant from cycle 3 wasidentified by growing colonies in microtiter plates and fluorescencespectrometry of the microtiter plates.

For characterization of mutants in E. coli, DNA sequencing was performedon an Applied Biosystems 391 DNA sequencer.

CHO cell expression of GFP

The wildtype and the cycle 2 and 3 mutant versions of the GFP gene weretransferred into the eukaryotic expression vector Alpha+ (16) as anEcoRI-XbaI fragment. The plasmids were transfected into CHO cells byelectroporation of 10⁷ cells in 0.8 ml with 40 μg of plasmed at 400 Vand 250 μF. Transformants were selected using 1 mg/ml G418 for 10-12days.

FACS analysis was carried out on a Becton Dickinson FACSTAR Plus usingan Argon ion laser tuned to 488 nm. Fluorescence was observed with a535/30 run bandpass filter.

RESULTS

Codon usage

E. coli expressing the synthetic GFP construct (`wt`) with altered codonusage yielded a nearly 3-fold greater whole cell fluorescence signalthan cells expressing the `Clontech` construct (FIG. 15A). Thecomparison was performed at full induction and at equal OD₆₀₀. Inaddition to the substitution of poor arginine codons in the `wt`construct and the N-terminal extension present in the `Clontech`construct, the expression vectors and GFP promoters are quite different.The cause of the improved fluorescence signal is not enhanced expressionlevel, it is improved protein performance.

Sexual PCR

The fluorescence signal of the synthetic `wt` GFP construct was furtherimproved by constructing a mutant library by sexual PCR methods asdescribed herein and in Stemmer WPC (1994) Proc. Natl. Acad. Sci.(U.S.A.) 91:10747 and Stemmer WPC (1994) Nature 370:389, incorporatedherein by reference, followed by plating and selection of the brightestcolonies. After the second cycle of sexual PCR and selection, a mutant(`cycle 2') was obtained that was about 8-fold improved over `wt`, and23-fold over the `Clontech` construct. After the third cycle a mutant(`cycle 3`) was obtained which was 16-18-fold improved over the `wt`construct, and 45-fold over the `Clontech` construct (FIG. 15B). Thepeak wavelengths of the excitation and emission spectra of the mutantswere identical to that of the `wt` construct (FIG. 15B). SDS-PAGEanalysis of whole ceils showed that the total level of the GFP proteinexpressed in all three constructs was unchanged, at a surprisingly highrate of about 75% of total protein (FIG. 16, panels (a) and (b)).Fractionation of the cells by sonication and centrifugation showed thatthe `wt` construct contained mostly inactive GFP in the form ofinclusion bodies, whereas the `cycle 3` mutant GFP remained mostlysoluble and was able to activate its chromophore. The mutant genes weresequenced and the `cycle 1` mutant was found to contain more mutationsthan the `cycle 3` mutant (FIG. 17). The `cycle 3` contained 3 proteinmutations and 3 silent mutations relative to the `wt` construct.Mutations F100S, M154T, and V164A involve the replacement of hydrophobicresidues with more hydrophilic residues (Kyte and Doolittle, 1982). Oneplausible explanation is that native GFP has a hydrophobic site on itssurface by which it normally binds to Aequorin, or to another protein.In the absence of this other protein, the hydrophobic site may causeaggregation and prevent autocatalytic activation of the chromophore. Thethree hydrophilic mutations may counteract the hydrophobic site,resulting in reduced aggregation and increased chromophore activation.Pulse chase experiments with whole bacteria at 37° C. showed that theT_(1/2) for fluorophore formation was 95 minutes for both the `wt` andthe `cycle 3` mutant GFP.

CHO cells

Improvements in autonomous characteristics such as self-folding can betranferable to different cellular environments. After being selected inbacteria, the `cycle 3` mutant GFP was transferred into the eukaryoticAlpha+vector and expressed in chinese hamster ovary cells (CHO). Whereasin E. coli the `cycle 3` construct gave a 16-18-fold stronger signalthan the `wt` construct, fluorescence spectroscopy of CHO cellsexpressing the `cycle 3` mutant showed a 42-fold greater whole cellfluorescence signal than the `wt` construct under identical conditions(FIG. 18A). FACS sorting confirmed that the average fluorescence signalof CHO cell clones expressing `cycle 3` was 46-fold greater than cellsexpressing the `wt` construct (FIG. 18B). As for the `wt` construct, theaddition of 2 mM sodium butyrate was found to increase the fluorescencesignal about 4-8 fold.

Screening versus selection

These results were obtained by visual screening of approximately 10,000colonies, and the brightest 40 colonies were picked at each cycle.Significant improvements in protein function can be obtained withrelatively low numbers of variants. In view of this surprising finding,sexual PCR can be combined with high throughput screening procedures asan improved process for the optimization of the large number ofcommercially important enzymes for which large scale mutant selectionsare not feasible or efficient.

Example 14. Shuffling to Generate Improved Peptide Display Libraries

Background

Once recombinants have been characterized from a phage display library,polysome display library, or the like, it is often useful to constructand screen a second generation library that displays variants of theoriginally displayed sequence(s). However, because the number ofcombinations for polypeptides longer than seven residues is so greatthat all permutations will not generally be present in the primarylibrary. Furthermore, by mutating sequences, the "sequence landscape"around the isolated sequence can be examined to find local optima.

There are several methods available to the experimenter for the purposesof mutagenesis. For example, suitable methods include site-directedmutagenesis, cassette mutagenesis, and error-prone PCR.

Overview

The disclosed method for generating mutations in vitro is known as DNAshuffling. In an embodiment of DNA shuffling, genes are broken intosmall, random fragments with DNase I, and then reassembled in a PCR-likereaction, but typically without any primers. The process of reassemblingcan be mutagenic in the absence of a proof-reading polymerase,generating up to about 0.7% error rate. These mutations consist of bothtransitions and transversion, often randomly distributed over the lengthof the reassembled segment.

Once one has isolated a phage-displayed recombinant with desirableproperties, it is generally appropriate to improve or alter the bindingproperties through a round of molecular evolution via DNA shuffling.Second generation libraries of displayed peptides and antibodies weregenerated and isolated phage with improved (i.e., 3-1000 fold) apparentbinding strength were produced. Thus, through repeated rounds of librarygeneration and selection it is possible to "hill-climb" through sequencespace to optimal binding.

From second generation libraries, very often stronger binding speciescan be isolated. Selective enrichment of such phage can be accomplishedby screening with lower target concentrations immobilized on amicroriter plate or in solution, combined with extensive washing or byother means known in the art. Another option is to display themutagenized population of molecules at a lower valency on phage toselect for molecules with higher affinity constants. Finally, it ispossible to screen second generation libraries in the presence of a lowconcentration of binding inhibitor (i.e., target, ligand) that blocksthe efficient binding of the parental phage.

Methods

Exemplary Mutagenesis Protocols

A form of recombinant DNA-based mutagenesis is known asoligonucleotide-mediated site-directed mutagenesis. An oligonucleotideis designed such that can it base-pair to a target DNA, while differingin one or more bases near the center of the oligonucleotide. When thisoligonucleotide is base-paired to the single-stranded template DNA, theheteroduplex is converted into double-stranded DNA in vitro; in thismanner one strand of the product will carry the nucleotide sequencespecific by the mutagenic oligonucleotide. These DNA molecules are thenpropagated in vivo and the desired recombinant is ultimately identifiedamong the population of transformants.

A protocol for single-stranded mutagenesis is described below.

1. Prepare single-stranded DNA from M13 phage or phagemids. Isolate.sup.˜ 2 μg of DNA. The DNA can be isolated from a dut⁻ ung⁻ bacterialhost (source) so that the recovered DNA contains uracil in place of manythymine residues.

2. Design an oligonucleotide that has at least 15 or 20 residues ofcomplementarity to the coding regions flanking the site to be mutated.In the oligonucleotide, the region to be randomized can be representedby degenerate codons. If the non-complementary region is large(i.e., >12 nucleotides), then the flanking regions should be extended toensure proper base pairing. The oligonucleotide should be synthesizedwith a 5'PO₄ group, as it improves the efficiency of the mutagenesisprocedure; this group can also be added enzymatically with T4polynucleotide kinase. (In an Eppendorf tube, incubate 100 ng ofoligonucleotide with 2 units of T4 polynucleotide kinase in 50 mM (pH7.5), 10 MM MgCI2,5 mM DTT, and OA mM ATP for 30 min.

3. Anneal the oligonucleotide with the single-stranded DNA in a 500 μlEppendorf tube containing: 1 μg single-stranded DNA, 10 ngoligonucleotide, 20 mM Tr@Cl (pH 7.4), 2 mM MgCl₂, 50 mM NaCl.

4. Mix the solutions together and centrifuge the tube for a few secondsto recollect the liquid. Heat the tube in a flask containing waterheated to 70° C. After 5 min, transfer the flask to the lab bench andlet it cool to room temperature slowly.

5. Take the tube out of the water bath and put it on ice. Add thefollowing reagents to the tube, for a total volume of 100 μl: 20 mMTris-HCI (pH 7.4), 2 mM DTT, 0.5 mM dATP, dCTP, dGTP and dTTP, 0.4 mMATP, 1 unit T7 DNA polymerase, 2 units T4 DNA ligase.

6. After 1 hr, add EDTA to 10 mM final concentration.

7. Take 20 μAl from the sample and run on an agarose gel. Most of thesingle-stranded DNA should be converted to covalently-closed circularDNA. Electrophorese some controls in adjacent lanes (i.e., template,template reaction without oligonucleotide). Add T4 DNA ligase to closethe double-stranded circular DNA.

8. Extract the remainder of the DNA (80 μl) by phenol extraction andrecover by ethanol precipitation.

9. Electroporate into ung⁺ bacteria.

10. Harvest the second generation phage by PEG precipitation.

Cassette mutagenesis

A convenient means of introducing mutations at a particular site withina coding region is by cassette mutagenesis. The "cassette" can begenerated several different ways: A) by annealing two oligonucleotidestogether and converting them into double stranded DNA; B) by firstamplifying segments of DNA with oligonucleotides that carry randomizedsequences and then reamplifying the DNA to create the cassette forcloning; C) by first amplifying each half of the DNA segment witholigonucleotides that carry randomized sequences, and then heating thetwo pieces together to create the cassette for cloning; and D) byerror-prone PCR. The cassettes formed by these four procedures are fixedin length and coding frame, but have codons which are unspecified at alow frequency. Thus, cloning and expression of the cassettes willgenerate a plurality of peptides or proteins that have one or moremutant residues along the entire length of the cassette.

Typically, two types of mutagenesis scheme can be used. First, certainresidues in a phage-displayed protein or peptide can be completelyrandomized. The codons at these positions can be NNN, NNK, or NNS whichuse 32 codons to encode all 20 residues. They can also be synthesized aspreformed triplets or by mixing oligonucleotides synthesized by thesplit-resin method which together cover all 20 codons at each desiredposition. Conversely, a subset of codons can be used to favor certainamino acids and exclude others. Second, all of the codons in thecassette can have some low probability of being mutated. This isaccomplished by synthesized oligonucleotides with bottles "spiked" withthe other three bases or by altering the ratio of oligonucleotides mixedtogether by the split-resin method.

For mutagenesis of short regions, cassette mutagenesis with syntheticoligonucleotide is generally preferred. More than one cassette can beused at a time to alter several regions simultaneously. This approach ispreferred when creating a library of mutant antibodies, where all sixcomplementarity determining regions (CDR) are altered concurrently.

Random codons

1. Design oligonucleotides with both fixed and mutated positions. Thefixed positions should correspond to the cloning sites and those codingregions presumed to be essential for binding or function.

2. During synthesis of the oligonucleotide, have the oligonucleotidesynthesizer deliver equimolar amounts of each base for N, guanosine andcytosine for K, guanosine and thymidine for S.

"Spiked" codons

1. Design oligonucleotides with both fixed and mutated positions. Thefixed positions should correspond to the cloning sites and those codingregions presumed to be essential for binding or function. Theprobability of finding n errors in an m long polynucleotide cassettesynthesized with x fraction of the other three nucleotides at eachposition is represented by:

    P=[m!/(m-n)n!][x.sup.n ] [1-x].sup.m-n

2. During synthesis of the oligonucleotide switch out the base bottles.Use bottles with 100% of each base for the fixed positions and bottlewith 100-x% of one base and x/3% of each of the other three bases. Thedoping ratio can also differ based on the average amino acid use innatural globular proteins or other algorithms. There is a commerciallyavailable computer program, CyberDope, which can be used to aid indetermining the base mixtures for synthesizing oligonudeotides withparticular doping schemes. A demonstration copy of the CyberDope programcan be obtained by sending an email request to cyberdope@aol.com.

Directed codons

1. Design oligonucleotides with both fixed and mutated positions. Thefixed positions should correspond to the cloning sites and those codingregions presumed to be essential for binding or function. One method hasbeen described for inserting a set of oligonucleotides at a specificrestriction enzyme site that encodes all twenty amino acids (Kegler-Eboet al. (1994) Nucl. Acids Res. 22:1593, incorporated herein byreference).

2. During synthesis of the oligonucleotide split the resin at eachcodon.

Error-prone PCR

There are several protocols based on altering standard PCR conditions(Saiki et al. (1988) Science 239:487, incorporated herein by reference)to elevate the level of mutation during amplification. Addition ofelevated dNTP concentrations and/or Mn⁺² increase the rate of mutationsignificantly. Since the mutations are theoretically introduced atrandom, this is one mechanism for generating populations of novelproteins. On the other hand, error-prone PCR is not well suited foraltering short peptide sequences because the coding regions are short,and the rate of change would be too low to generate an adequate numberof mutants for selection, nor is it ideal for long proteins, becausethere will be many mutations within the coding region which complicatesanalysis.

1. Design oligonucleotide primers that flank the coding region ofinterest in the phage. They are often approximately 21 nucleotides inlength and flank the region to be mutagenized. The fragment to beamplified can carry restriction sites within it to permit easysubcloning in the appropriate vector.

2. The following reaction is set up:

1 pmole of each primer; 1 pmole of the DNA template; 100 mM NaCl, 1 mMMnCl₂, 1 mM DTT, 0.2 mM of each dNTP, 2 units of Taq DNA polymerase.

3. Cover the liquid with mineral oil.

4. Cycle 24 times between 30 sec at 94° C., 30 sec 45° C., and 30 sec at72° C. to amplify fragments up to 1 kb. For longer fragments, the 72° C.step is lengthened by approximately 30 sec for each kb.

5. Extend the PCR reaction for 5-10 min at 72° C. to increase thefraction of molecules that are full-length. This is important if thefragment termini contain restriction sites that will be used insubcloning later.

6. The PCR reaction is optionally monitored by gel electrophoresis.

7. The PCR product is digested with the appropriate restrictionenzyme(s) to generate sticky ends. The restriction fragments can be gelpurified.

8. The DNA segment is cloned into a suitable vector by ligation andintroduced into host cells.

DNA Shuffling

In DNA shuffling, genes are broken into small, random fragments with aphosphodiester bond lytic agent, such as DNase I, and then reassembledin a PCR-like reaction, but without requirement for any added primers.The process of reassembling can be mutagenic in the absence of aproof-reading polymerase, generating up to approximately 0.7% error when10-50 bp fragments are used.

1. PCR amplify the fragment to be shuffled. Often it is convenient toPCR from a bacterial colony or plaque. Touch the colony or plaque with asterile toothpick and swirl in a PCR reaction mix (buffer,deoxynucleotides, oligonucleotide primers). Remove the toothpick andbeat the reaction for 10 min at 99° C. Cool the reaction to 72° C., add1-2 units of Taq DNA polymerase, and cycle the reaction 35 times for 30sec at 94° C., 30 sec at 45° C., 30 sec at 72° C. and finally heat thesample for 5 min at 72° C. (Given conditions are for a 1 kb gene and aremodified according the the length of the sequence as described.)

2. Remove the free primers. Complete primer removal is important.

3. Approximately 2-4 μg of the DNA is fragmented with 0.15 units ofDNase I (Sigma, St. Louis, Mo.) in 100 Al of 50 mM Tris-HCl (pH 7.4), 1mM MgCl₂, for 5-10 min at room temperature. Freeze on dry ice, checksize range of fragments on 2% low melting point agarose gel orequivalent, and thaw to continue digestion until desired size range isused. The desired size range depends on the application; for shufflingof a 1 kb gene, fragments of 100-300 bases are normally adequate.

4. The desired DNA fragment size range is gel purified from a 2% lowmelting point agarose gel or equivalent. A preferred method is to inserta small piece of Whatman DE-81 ion-exchange paper just in front of theDNA, run the DNA into the paper, put the paper in 0.5 ml 1.2 M NaCl inTE, vortex 30 sec, then carefully spin out all the paper, transfer thesupematant and add 2 volumes of 100% ethanol to precipitate the DNA; nocooling of the sample should be necessary. The DNA pellet is then washedwith 70% ethanol to remove traces of salt.

5. The DNA pellet is resuspended in PCR mix (Promega, Madison, Wis.)containing 0.2 mM each DNTP, 2.2 mM MgCl₂,50 mM KCl, 10 mM Tris-HCl, pH9.0, 0.1% Triton X100, at a concentration of about 10-30 ng of fragmentsper μl of PCR mix (typically 100-600 ng per 10-20 Al PCR reaction).Primers are not required to be added in this PCR reaction. Taq DNApolymerase (Promega, Madison, Wis.) alone can be used if a substantialrate of mutagenesis (up to 0.7% with 10-50 bp DNA fragments) is desired.The inclusion of a proof-reading polymerase, such as a 1:30 (vol/vol)mixture of Taq and Pfu DNA polymerase (Stratagene, San Diego, Calif.) isexpected to yield a lower error rate and allows the PCR of very longsequences. A program of 30-45 cycles of 30 sec 94° C., 30 sec 45-50° C.,30 sec 72° C., hold at 4° C. is used in an MJ Research PTC-150minicycler (Cambridge, Mass.). The progress of the assembly can bechecked by gel analysis. The PCR product at this point contains thecorrect size product in a smear of larger and smaller sizes.

6. The correctly reassembled product of this first PCR is amplified in asecond PCR reaction which contains outside primers. Aliquots of 7.5 μlof the PCR reassembly are diluted 40× with PCR mix containing 0.8 pM ofeach primer. A PCR program of 20 cycles of 30 sec 94° C., 30 sec 50° C.,and 30-45 sec at 72° C. is run, with 5 min at 72° C. at the end.

7. The desired PCR product is then digested with terminal restrictionenzymes, gel purified, and cloned back into a vector, which is oftenintroduced into a host cell.

Site-specific recombination can also be used, for example, to shuffleheavy and light antibody chains inside infected bacterial cells as ameans of increasing the binding affinity and specificity of antibodymolecules. It is possible to use the Cre/lox system (Waterhouse et al.(1993) Nucl. Acids Res. 21:2265; Griffiths et al. (1994) EMBO J.13:3245, incorporated by reference) and the int system.

It is possible to take recombinants and to shuffle them together tocombine advantageous mutations that occur on different DNA molecules andit is also possible to take a recombinant displayed insert and to"backcross" with parental sequences by DNA shuffling to remove anymutations that do not contribute to the desired traits.

Example 15. Shuffling to Generate Improved Arsenate DetoxificationBacteria

Arsenic detoxification is important for goldmining of arsenopyritecontaining gold ores and other uses, such as environmental remediation.Plasmid pGJ103, containing an operon encoding arsenate detoxificationoperon (Wang et al. (1989) Bacteriol. 171:83, incorporated herein byreference), was obtained from Prof. Simon Silver (U. of Illinois,Chicago, Ill.). E. coli TG1 containing pJG103, containing the p1258 arsoperon cloned into pUC19, had a MIC (minimum inhibitory concentration)of 4 μg/ml on LB amp plates. The whole 5.5 kb plasmid was fragmentedwith DNAse I into fragments of 100-1000 bp, and reassembled by PCR usingthe Perkin Elmer XL-PCR reagents. After assembling, the plasmid wasdigested with the unique restriction enzyme BamHI. The full lengthmonomer was purified from the agarose gel, ligated and electroporatedinto E. coli TG1 cells. The tranformed cells were plates on a range ofsodium arsenate concentrations (2, 4, 8, 16 mM in round 1), and approx.1000 colonies from the plates with the highest arsenate levels werepooled by scraping the plates. The cells were grown in liquid in thepresence of the same concentration of arsenate, and plasmid was preparedfrom this culture. Round 2 and 3 were identical to round 1, except thatthe cells were plated at higher arsenate levels. 8, 16, 32, 64 mM wereused for round 2; and 32, 64, 128, 256 mM were used for selection ofround 3.

The best mutants grew overnight at up to 128 mM arsenate (MIC=256), a64-fold improvement. One of the improved strains showed that the TG1(wildtype pGGJ103) grew in liquid at up to 10 mM, whereas the shuffledTG1(mutant pGJ103) grew at up to 150 mM arsenate concentration.

PCR program for the assembly was 94° C. 20s, 50×(94° C. 15s, 50° C. 1min, 72° C. 30s+2s/cycle), using a circular PCR format without primers.

Four cycles of the process resulted in a 50-100-fold improvement in theresistance to arsenate conferred by the shuffled arsenate resistanceoperon; bacteria containing the improved operon grew on mediumcontaining up to 500 mM arsenate.

FIG. 19 shows enhancement of resistance to arsenate toxicity as a resultof shuffling the pGJ103 plasmid containing the arsenate detoxificationpathway operon.

Example 16. Shuffling to Generate Improved Cadmium DetoxificationBacteria

Plasmid pYW333, containing an operon for mercury detoxification is a15.5 kb plasmid containing at least 8 genes encoding a pathway formercury detoxification (Wang et al. (1989) Bacteriol. 171:83,incorporated herein by reference), was obtained from Prof. Simon Silver(Univ. Illinois, Chicago, Ill.). 400-1500 bp fragments were obtained asdescribed supra and assembled with the XL-PCR reagents. After directelectroporation of the assembled DNA into to E. Coli TG1, the cells wereplated on a range of levels of mercury chloride (Sigma) under a similarprotocol as that described for arsenate in Example 15. The initial MICof mercury was 50-70 μM. Four cycles of whole plasmid shuffling wereperformed and increased the detoxification measured as bacterialresistance to mercury from about 50-70 μM to over 1000 μM, a 15-20 foldimprovement.

Example 17. Enhancement of Shuffling Reactions by Addition of CationicDeterrent

The rate of renaturation of complementary DNA strands becomes limitingfor the shuffling long, complex sequences. This renaturation rate can beenhanced 10,000-fold by addition of simple cationic detergent (Pontiusand Berg (1991) PNAS 88:8237). The renaturation is specific andindependent of up to a 10⁶ -fold excess of heterologous DNA. In thepresence of these agents the rate which the complementary DNA standsencounter each other in solution becomes limiting.

Addition of TMAC in an assembly reaction of a 15 kb plasmid followed byelectroporation into E. coli resulted in the following results:

    ______________________________________                                               TMAC (mM)                                                                              # Colonies                                                    ______________________________________                                               0        3                                                               15 88                                                                         30 301                                                                        60 15                                                                         90 3                                                                        ______________________________________                                    

Addition of CTAB in an assembly reaction of a 15 kb plasmid followed byelectroporation into E. coli resulted in the following results:

    ______________________________________                                               CTAB (mM)                                                                             # Colonies                                                     ______________________________________                                                       154                                                              0 3                                                                           30 34                                                                         100 14                                                                        300 0                                                                       ______________________________________                                    

Example 18. Sequence Shuffling via PCR Stuttering

Stuttering is fragmentation by incomplete polymerase extension oftemplates. A recombination format based on very short PCR extensiontimes was employed to create partial PCR products, which continue toextend off a different template in the next (and subsequent) cycle(s).There was a strong growth rate bias between very similar templates,indicating that this format has significant limitations for theapplication to complex pools. If used with a rapid cycler such as theaircycler, this format may work better.

PCR programs used for PCR stuttering were 100 cycles of (94° C. 15 sec,60° C. 15 sec). Two separate PCR reactions were run to obtain 1 kb PCRfragments containing the each of two GFP-negative recombination assaysubstrates (GFPstop1 and GFP stop2). GFP-positive recombinants can onlybe obtained by recombination of these two templates. The oligonucleotideprimers used at the 5' end is 5'TAGCGGATCCTACCTACCTGACGC, containing anNheI site, and the oligo at the 3' end is 5'GAAAATCTTCTCTCATCC,containing an EcoRI site. A PCR reaction was set up with a 1ng of eachGFP-negative gene as a template. Taq PCR reagents can be used, but theuse of error-prone PCR conditions (Leung, 1989; Cadwell and Joyce, 1992)which reduce the processivity, can increase the percentage ofGFP-positive recombinants.

A stuttering program of 50-150 cycles of 94° C. 10-20s, 60° C. 10-30swas used on a Stratagene Robocycler. The stuttered PCR product wasdigested with NheI and Eco RI and cloned back into pBAD18 vectordigested with NheI and EcoRI and electroporated into E. coli and platedon amp plates. GFP-positive colonies arise by recombination between thetwo gFP-negative DNA sequences and were detected. The percentage ofGFP-positive colonies obtained by stuttering was between 0.1-10%,depending on conditions.

A synthetic gene was designed for each protein, using the identical(optimal E. coli) codon usage based on the native amino acid sequence.This approach increases the DNA homology of the synthetic genes relativeto the naturally occurring genes, and allows us to shuffle moredistantly related sequences than would be possible without the codonusage adjustment. Each of the four genes was assembled from 30 60 meroligos and 6 40 mers. The assembly was performed by assembly PCR asdescribed by Stemmer et al (1995; Gene 164:49-53). After assembling, thegenes were cloned into Sfi 1 sites of the vector pUC322 Sfi-BLA-Sfi(Stemmer et al (1995) Gene 164:49-53), and plated on a selective media.The minimum inhibitory activity of these four constructs for a widevariety of betalactam antibiotics was established. Cefotaxime was one ofthe antibiotics that was selected for optimization against. The fourgenes were shuffled by pooling 1 ug of the CR product of each gene,followed by DNAseI digestion of the pool and purification of 100-300 bpfragments from agarose gels. The purified fragments were reassembled bysexual PCR initially without outside primers, and then the full-lengthproduct was amplified in the presence of the outside primers. Theresulting full-length genes were digested with SfiI and ligated intofresh pUC322Sfi-Sfi vector and electroporated into fresh E. coli cells,and plated on increasing concentration of several antibiotics, includingcefotaxime, as described previously by Stemmer (1994) Nature370:389-391.

A manual shuffling PCR protocol is preferred for the mixing of genesthat are less than 80-90% homologous. The manual PCR uses a heat-labileDNA polymerase, such as DNA poll Klenow fragment. The initial PCRprogram with fragments in Klenow buffer at 10-30 ng/ul and dNTPs:

1-Denature 94° C. 20s

2-Quick-cool: dry ice ethanol 5s, ice 15s

3-Add Klenow enzyme

4-Anneal/extend 2 min 25° C.

5-cycle back to denature (cycle 1)

This is repeated for 10-20 cycles to initiate the template switching,after which regular PCR with heatstable polymerases is continued for anadditional 10-20 cycles to amplify the amount of product.

Example 19. Shuffling of Antibody Phage Display Libraries

A stable and well-expressed human single-chain Fv framework (V_(H)251-V_(L) A25) was obtained from an Ab-phage library constructed fromnaive human mRNA by selection for binding to diptheria toxin. This scFvframework was used to construct a naive Ab-phage library containing sixsynthetically mutated CDRs based on the germline sequences. The degreeof mutagenesis of each residue was similar to its naturally occurringvariability within its V-region family.

A PCR product containing the scFv gene was randomly fragmented withDNaseI digestion and fragments of 50-100 bp were purified. Syntheticoligonucleotides, each containing a mutated CDR flanked by 19 bp ofhomology to the scFv template, were added to the random fragments at a10:1 molar ratio. A library of full length, mutated scFV genes wasreassembled from the fragments by sexual PCR. Cloning into the pIIIprotein of M13 phage yielded an Ab-phage library of 4'10⁷ plaque-formingunits. The combinations of mutant and native CDRs were characterized bycolony PCR with primers specific for the native CDRs (see, FIG. 7). Allsix mutated CDRs were incorporated with 32-65% efficiency and a widevariety of combinations. Sequencing of the mutated CDRs showed that theobserved mutation rate matched the expected rate.

This Ab-phage library was panned for two rounds in microtiter plates forbinding to ten human protein targets, and seven of these targets yieldedELISA-positive clones. One target which resulted in positive clones wasthe human G-CSF receptor. The G-CSF receptor positive clones weresubjected to a second round and one quarter of second round phage cloneswere ELISA-positive for binding to the G-CSF receptor.

This diverse pool was used to evaluate the suitability of threedifferent sequence optimization strategies (conventional PCR,error-prone PCR, and DNA shuffling). After a single cycle of each ofthese alternatives, the DNA shuffling showed a seven-fold advantage,both in the percentage of Ab-phage recovered and in the G-CSFreceptor-specific ELISA signal. The panning was continued for sixadditional cycles, shuffling the pool of scFv genes after each round.The stringency of selection was gradually increased to two one-hourwashes at 50° C. in PBS-Tween in the presence of excess soluble G-CSFreceptor. In rounds 3 to 8, nearly 100 percent of the clones were ELISApositive. When pools from different cycles were assayed at identicalstringency, the percentage of phage bound increased 440-fold from cycle2 to cycle 8, as shown in FIG. 31. Individual phage clones from eachround showed a similar increase in specific ELISA signal. Sequencingshowed that the scFv contained an average of 34 (n=4) amino acidmutations, of which only four were present in all sequences evaluated.

In order to reduce potential immunogenicity, neutral or weaklycontributing mutations were removed by two cycles of backcrossing, witha 40-fold excess of a synthetically constructed germline scFv gene,followed by stringent panning. The average number of amino acidmutations in the backcrossed scFvs were nearly halved to 18 (n=3), ofwhich only four were present in all sequences. The backcrossed Ab phageclones were shown to bind strongly and with excellent specificity to thehuman G-CSF receptor. FIG. 32 shows the effect of ten selection roundsfor several human protein targets; six rounds of shuffling and tworounds of backcrossing were conducted. FIG. 33 shows the relativerecovery rates of phage, by panning with BSA, Ab 179, or G-CSF receptor,after conventional PCR ("non-shuffled"), error-prone PCR, or recursivesequence recombination ("shuffled").

Example 20. Optimization of GFP in Mammalian Cells

The plasmid vector pCMV-GFP, which encodes GFP and expresses it underthe control of a CMV promoter, was grown in TG1 cells and used totransfect CHO cells for transient expression assays.

Plasmid was rescued from FACS selected transiently expressing TG1 cellsby a proteinase K method or a PreTaq method (Gibco/BRL). Basically, theFACS collected cells were pelleted by centrifugation, freeze-thawedrepetitively, incubated with either proteinase K or PreTaq,phenol/chloroform extracted, ethanol precipitated, used to transform E.coli which were then plated on Amp plates. The results were:

Proteinase K method: input--5×10⁴ rescued 3×10⁴

PreTaq method: input--5×10⁴ rescued 2×10⁴

The rescued plasmid was grown up and 5 μg was partially digested withDNAseI and 50 to 700 bp fragments were gel purified, ethanolprecipitated, and resuspended in 330μl of 3.3x PCR buffer, 44 μlMg(OAc)₂ (Perkin-Elmer), 193 μl 40% PEG, 80 μl 10 mM dNTPs, 20 μl Tthpolymerase, 2 μl Pfu polymerase, 7 Al TMAC (Sigma), and 367 μl H₂ O. PCRwas conducted on a MJ Research PTC-150 minicycler for 40 cycles (94° C.,30 sec; 50° C., 30 sec, 72° C., 60 sec) with three sets of primers,which yielded three end-overlapping PCR fragments, which together andafter digestion and ligation reconstituted the entire plasmid. The PCRfragments were digested with AlwN1 and the fragments were gel purified,ligated, and electroporated into TG1 cells. Plasmid DNA was prepared andelectroporated into CHO cells, which were screened by FACS for the cellstransiently expressing the brightest GFP signals.

While the present invention has been described with reference to whatare considered to be the preferred examples, it is to be understood thatthe invention is not limited to the disclosed examples. To the contrary,the invention is intended to cover various modifications and equivalentarrangements included within the spirit and scope of the appendedclaims.

REFERENCES

The following references are cited in this application at the relevantportion of the application.

1. Holland, J. H. (1992) Sci. Am. July, 66-72.

2. Holland, J. H. (1992) "Adaptation in natural and artificial systems".Second edition, MIT Press, Cambridge.

3. Joyce, G. F. (1992) Scientific American, December, 90-97.

4. Kauffman, S. A. (1993) "The origins of order". Oxford UniversityPress, New York.

5. Stormo, G. D. (1991) Methods Enzymol. 208:458-468.

6. Schneider, T. D. et al., (1986) J. Mol. Biol. 188:415-431.

7. Reidhaar-Olson, J. F and Sauer, R. T. (1988) Science 241:53-57.

8. Stemmer, W. P. C. et al., (1992) Biotechniques 14:256-265.

9. Yockey, H. P. (1977) J. Theor. Biol. 67:345-376.

10. Yockey, H. P. (1974) J. Theor. Biol. 46:369-380.

11. Leung, D. W. et al., (1989) Technique 1:11-15.

12. Caldwell, R. C. and Joyce, G. F. (1992) PCR Methods and Applications2:28-33.

13. Bartel, D. P., and Szostak, J. W. (1993) Science 261:1411-1418.

14. Bock, L. C. et al., (1992) Nature 355:564-566.

15. Scott, J. K. and Smith, G. P. (1990) Science 249:386-390.

16. Cwirla, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-6382.

17. McCafferty, J. et al. (1990) Nature 348:552-554.

18. Cull, M. G. et al., (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869.

19. Gramm, H. et al., (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580.

20. Arkin, A. and Youvan, D. C. (1992) Proc. Natl. Acad. Sci. USA89:7811-7815.

21. Oliphant, A. R. et al., (1986) Gene 44:177-183.

22. Hermes, J. D. et al., (1990) Proc. Natl. Acad. Sci. USA 87:696-700.

23. Meyerhans, A. et al., (1990) Nucleic Acids Res. 18:1687-1691.

24. Osterhout, J. J. et al., (1992) J. Am. Chem. Soc. 114:331-337.

25. Cano, R. J. et al., (1993) Nature 363:536-538.

26. Palzkill and Botstein, (1992) J. Bacteriol. 174:5237-5243.

27. Marton et al., Nucleic Acids Res. 19:2423.

28. Yanish-Perron et al., [1985] Gene 33:103-119.

29. Watson (1988) Gene 70:399-403.

30. Ambler et al. (1991) Biochem J. 276:269-272.

31. Chen and Clowes, (1984) Nucleic Acid Res. 12:3219-3234.

32. Witholt, B. ([1987] Anal. Biochem. 164(2):320-330

33. Kabat et al., (1991) "Sequences of Proteins of ImmunologicalInterest" U.S. Department of Health and Human Services, NIH Publication91-3242.

34. Philippon et al., (1989) Antimicrob Agents Chemother 33:1131-1136.

35. Jacoby and Medeiros (1991) Antimicrob. Agents Chemother.35:167-1704.

36. Coelhosampaio (1993) Biochem. 32:10929-10935

37. Tuerk, C. et al., (1992) Proc. Natl. Acad. Sci. USA 89:6988-6992.

38. U.S. Pat. No. 4,683,195

39. U.S. Pat. No. 4,683,202

40. Delagrave et al. (1993) Protein Engineering 6:327-331

41. Delgrave et al. (1993) Bio/Technology 11:1548-1552

42. Goldman, E. R. and Youvan D. C. (1992) Bio/Technology 10:1557-1561

43. Nissim et al. (1994) EMBO J. 13:692-698

44. Winter et al. (1994) Ann. Rev. Immunol. 12:433-55

45. Caren et al. (1994) Bio/Technology 12:517-520

46. Calogero et al. (1992) FEMS Microbiology Lett. 97:41-44

47. Galizzi et al. WO91/01087

48. Hayashi et al. (1994) Biotechniques 17:310-315

49. Radman et al. WO90/07576

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. PCT/US95/02126 filed Feb. 17, 1995 and published onAug. 24, 1995 as WO95/22625 is incorporated herein by reference.Copending application attorney docket no. 16528A-0196 is incorporatedherein by reference.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 70                                          - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - AAAGCGTCGA TTTTTGTGAT            - #                  - #                      - # 20                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - ATGGGGTTCC GCGCACATTT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:3:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                               - - TTAGGCACCC CAGGCTTT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:4:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                               - - ATGTGCTGCA AGGCGATT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:5:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                               - - AACGCCGCAT GCAAGCTTGG ATCCTTATT         - #                  - #                29                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:6:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                               - - AAAGCCCTCT AGATGATTAC GAATTCATAT         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:7:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                               - - TTCTATTGAC GGCCTGTCAG GCCTCATATA TACTTTAGAT TGATTT   - #                     46                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:8:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                               - - TTGACGCACT GGCCATGGTG GCCAAAAATA AACAAATAGG GGTTCCGCGC AC - #ATTT             56                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:9:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                               - - AACTGACCAC GGCCTGACAG GCCGGTCTGA CAGTTACCAA TGCTT   - #                      - #45                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:10:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                              - - AACCTGTCCT GGCCACCATG GCCTAAATAC ATTCAAATAT GTAT   - #                      - # 44                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:11:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 31 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                              - - AGTTGGGTGG ACGAGTGGGT TACATCGAAC T        - #                  - #              31                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:12:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 33 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                              - - AACCCACTCG TCCACCCAAC TGATCTTCAG CAT       - #                  - #             33                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:13:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                              - - AGTAAAAGAT GCTGAAGATA AGTTGGGTGC ACGAGTGGGT T    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:14:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                              - - ACTTATCTTC AGCATCTTTT ACTT          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:15:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 32 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                              - - AAGAGCAACT CAGTCGCCGC ATACACTATT CT       - #                  - #              32                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:16:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                              - - ATGGCGGCGA CTGAGTTGCT CTTGCCCGGC GTCAAT      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:17:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                              - - TATTCTCAGA ATGACTTGGT TAAGTACTCA CCAGTCACAG AA    - #                      - #  42                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:18:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                              - - TTAACCAAGT CATTCTGAGA AT           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:19:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                              - - AACGACGAGC GTGACACCAC GACGCCTGTA GCAATG      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:20:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                              - - TCGTGGTGTC ACGCTCGTCG TT           - #                  - #                     22                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:21:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 42 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                              - - TTGCTGATAA ATCTGGAGCC AGTGAGCGTG GGTCTCGCGG TA    - #                      - #  42                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:22:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                              - - TGGCTCCAGA TTTATCAGCA A           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:23:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                              - - ATGCTCACTG GCTCCAGATT TATCAGCAAT         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:24:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 36 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                              - - TCTGGAGCCA GTGAGCATGG GTCTCGCGGT ATCATT      - #                  -     #       36                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:25:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                              - - AACCTGTCCT GGCCACCATG GCCTAAATAC AATCAAATAT GTATCCGCTT AT -             #GAGACAAT     60                                                                 - - AACCCTGATA                - #                  - #                      - #        70                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:26:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                              - - ATGATTACGC CAAGCTTT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:27:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                              - - TTGTCGTCTT TCCAGACGTT            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:28:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                              - - TTCTGGCTAC ATCTTCACAA CTTATGATAT AGACTGGGTG AGGCAGACGC CT - #GAA              55                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:29:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 91 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                              - - ACAGGGACTT GAGTGGATTG GATGGATTTT TCCTGGAGAG GGTGGTACTG AA -             #TACAATGA     60                                                                 - - GAAGTTCAAG GGCAGGGCCA CACTGAGTGT A        - #                  - #              91                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:30:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 71 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                              - - TGTCTATTTC TGTGCTAGAG GGGACTACTA TAGGCGCTAC TTTGACTTGT GG -             #GGCCAAGG     60                                                                 - - GACCACGGTC A               - #                  - #                      - #       71                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:31:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                              - - AGAGGGTCAC CATGACCTGC AGTGCCAGCT CAGGTATACG TTACATATAT TG -             #GTACCAAC     60                                                                 - - AGAAGCCTGG AT              - #                  - #                      - #       72                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:32:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                              - - TCCCCCAGAC TCCTGATTTA TGACACATCC AACGTGGCTC CTGGAGTCCC TT -             #TTCGCTTC     60                                                                 - - AGT                  - #                  - #                  - #                 63                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:33:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 68 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                              - - ATGCTGCCAC TTATTACTTG CCAGGAGTGG AGTGGTTATC CGTACACGTT CG -             #GAGGGGGG     60                                                                 - - ACCAAGCT                - #                  - #                       - #          68                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:34:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 55 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                              - - TTCTGGCTAC ATCTTCACAG AATTCATCTA GATTTGGGTG AGGCAGACGC CT - #GAA              55                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO:35:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 91 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                              - - ACAGGGACTT GAGTGGATTG GAATCACAGT CAAGCTTATC CTTTATCTCA GG -             #TCTCGAGT     60                                                                 - - TCCAAGTACT TAAAGGGCCA CACTGAGTGT A        - #                  - #              91                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:36:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 70 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                              - - TGTCTATTTC TGTGCTAGAT CTTGACTGCA GTCTTATACG AGGATCCATT GG -             #GGCCAAGG     60                                                                 - - GACCAGGTCA                - #                  - #                      - #        70                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:37:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 72 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                              - - AGAGGGTCAC CATGACCTGC GGACGTCTTT AAGCGATCGG GCTGATGGCC TG -             #GTACCAAC     60                                                                 - - AGAAGCCTGG AT              - #                  - #                      - #       72                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:38:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 63 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                              - - TCCCCCAGAC TCCTGATTTA TTAAGGGAGA TCTAAACAGC TGTTGGTCCC TT -             #TTCGCTTC     60                                                                 - - AGT                  - #                  - #                  - #                 63                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:39:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 67 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                              - - ATGCTGCCAC TTATTACTGC TTCTGCGCGC TTAAAGGATA TCTTCATTTC GG -             #AGGGGGGA     60                                                                 - - CCAAGCT                 - #                  - #                       - #          67                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:40:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                              - - AGAATTCATC TAGATTTG             - #                  - #                      - #  18                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:41:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                              - - GCTTATCCTT TATCTCAGGT C           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:42:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                              - - ACTGCAGTCT TATACGAGGA T           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:43:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                              - - GACGTCTTTA AGCGATCG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:44:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                              - - TAAGGGAGAT CTAAACAG             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:45:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                              - - TCTGCGCGCT TAAAGGAT             - #                  - #                      - #  18                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:46:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 46 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                              - - TTCTATTGAC GGCCTGTCAG GCCTCATATA TACTTTAGAT TGATTT   - #                     46                                                                         - -  - - (2) INFORMATION FOR SEQ ID NO:47:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 56 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                              - - TTGACGCACT GGCCATGGTG GCCAAAAATA AACAAATAGG GGTTCCGCGC AC - #ATTT             56                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:48:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 45 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                              - - AACTGACCAC GGCCTGACAG GCCGGTCTGA CAGTTACCAA TGCTT   - #                      - #45                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:49:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 44 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                              - - AACCTGTCCT GGCCACCATG GCCTAAATAC ATTCAAATAT GTAT   - #                      - # 44                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:50:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                              - - TTCTTAGACG TCAGGTGGCA CTT           - #                  - #                    23                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:51:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                              - - TTTTAAATCA ATCTAAAGTA T           - #                  - #                      - #21                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:52:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 41 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                              - - TGCTCATCCA CGAGTGTGGA GAAGTGGTCC TGCAACTTTA T    - #                      - #   41                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:53:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 39 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                              - - ACCACTTCTC CACACTCGTG GATGAGCACT TTTAAAGTT      - #                      - #    39                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:54:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 53 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                              - - TGTCACTACC ACACTCGTGG ACTACCATGG CCTAAATACA TTCAAATATG TA - #T                53                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:55:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                              - - TTAAGGGATT TTGGTCATGA GATT          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:56:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                              - - TAGCGGATCC TACCTGACGC            - #                  - #                      - # 20                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:57:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                              - - GAAAATCTTC TCTCATCCG             - #                  - #                      - # 19                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO:58:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 81 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                              - - GTCGACCTCG AGCCATGGCT AACTAATTAA GTAATTACTG CAGCGTCGTG AC -             #TGGGAAAA     60                                                                 - - CCCTGGGGTT ACCCAACTTA A           - #                  - #                      - #81                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO:59:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 84 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                              - - GTCGACCTGC AGGCATGCAA GCTTAGCACT TGCTGTAGTA CTGCAGCGTC GT -             #GACTGGGA     60                                                                 - - AAACCCTGGG GTTACCCAAC TTAA          - #                  - #                    84                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:60:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                              - - TCGCCTTGCT GCGCATCCAC CTTTCGCTAG CTGGCGGAAT TCCGAAGAAG CG - #CG               54                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:61:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 57 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                              - - TCGCCTTGCT GCGCATCCAC CTTTCGCTAG TTAACTAATT AACTAAGATA TC - #GCGCG            57                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:62:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 462 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                              - - ATGGTTCCGA TCCGTCAGCT GCACTACCGT CTGCGTGACG AACAGCAGAA AA -             #GCCTGGTT     60                                                                 - - CTGTCCGACC CGTACGAACT GAAAGCTAGG TGATCTTCTC CATGAGCTTC GT -            #ACAAGGTG    120                                                                 - - AACCAAGCAA CGACAAAATC CCGGTGGCTT TGGGTCTGAA AGGTAAAAAC CT -            #GTGACCCT    180                                                                 - - GCAACTCGAG AGCGTGGACC CAAAACAGTA CCCAAAGAAG AAGATGGAGA AG -            #CGTTTCGT    240                                                                 - - CTTCAACAAG ATCGAAGTCA ACCGAACTGG TACATCAGCA CCTCCCAAGC AG -            #AGCACAAG    300                                                                 - - CCTGTCTTCC TGGGTAACAA CTCCGGTCAG GATATCATCG ACTTCCTGCA CC -            #TGAATGGC    360                                                                 - - CAGAACATCA ACCAACACCT GTCCTGTGTA ATGAAAGACG GCACTCCGAG CA -            #AAGTGGAG    420                                                                 - - TTCGAGTCTG CTGAGTTCAC TATGGAATCT GTGTCTTCCT AA    - #                      - # 462                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO:63:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 465 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA (genomic)                                     - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                              - - ATGGCACCGG TTAGATCTCT GAACTGCACC CTTCGCGACT CCCAACAGAA AA -             #GCTTAGTA     60                                                                 - - ATGTCTGGTC CGTACGAGCT CAAAGCTAGG TTGTATTCAG CATGAGCTTC GT -            #CCAAGGTG    120                                                                 - - AAGAGTCTAA CGACAAGATC CCAGTTGCAT TAGGCCTGAA AGAGAAGAAT CT -            #GTGACTCT    180                                                                 - - GCAGCTTGAA TCCGTTGACC CGAAAAACTA TCCGAAGAAG AAAATGGAGA AG -            #CGTTTCGT    240                                                                 - - ATTTAACAAG ATTGAGATTA ACCAAACTGG TACATCAGTA CTTCTCAAGC AG -            #AGAATATG    300                                                                 - - CCTGTGTTCC TCGGCGGTAC CAAAGGCGGT CAGGATATCA CTGACTTCCT GC -            #ATCTGCAA    360                                                                 - - GGCCAGCACA TGGAACAACA CCTCAGCTGC GTACTGAAAG ACGATAAGCC TA -            #ACAAGCTG    420                                                                 - - GAATTCGAGT CTGCTCAGTT CACCATGCAG TTTGTCTCGA GCTAA   - #                     465                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:64:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino - #acids                                                  (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                              - - Gly Gly Gly Gly Ser                                                      1               5                                                              - -  - - (2) INFORMATION FOR SEQ ID NO:65:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                              - - NNKNNKNNKN NKNNKNNKNN KNNKNNKNNK         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:66:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 30 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                              - - NNMNNMNNMN NMNNMNNMNN MNNMNNMNNM         - #                  - #               30                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:67:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 amino - #acids                                                 (B) TYPE: amino acid                                                          (C) STRANDEDNESS:                                                             (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: peptide                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                              - - Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gl - #y Gly Gly Gly Ser          1               5   - #                10  - #                15               - -  - - (2) INFORMATION FOR SEQ ID NO:68:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 54 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                              - - ATGGTAGTCC ACGAGTGTGG TAGTGACAGG CCGGTCTGAC AGTTACCAAT GC - #TT               54                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO:69:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                              - - TAGCGGATCC TACCTACCTG ACGC          - #                  - #                    24                                                                      - -  - - (2) INFORMATION FOR SEQ ID NO:70:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base - #pairs                                                  (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: DNA                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                              - - GAAAATCTTC TCTCATCC             - #                  - #                      - #  18                                                                 __________________________________________________________________________

What is claimed is:
 1. A method for producing a variant polynucleotide,comprising:(a) conducting a polynucleotide extension process underconditions resulting in incomplete extension of synthesizedpolynucleotides to produce a plurality of polynucleotides in variousstages of synthesis wherein at least 20% of the amplified strands areincompletely extended; (b) denaturing the plurality of polynucleotides;(c) incubating the denatured plurality of polynucleotides with apolymerase under conditions which result in annealing of single-strandedpolynucleotides at regions of complementarity between the singlestranded polynucleotides and formation of variant double strandedpolynucleotide chains; (d) repeating (b) and (c); (e) screening orselecting a plurality of variant polynucleotides or expression productsthereof to identify a variant polynucleotide having a useful functionalactivity.
 2. The method of claim 1, wherein the conditions resulting inincomplete extension are achieved by shortening the extension time ofthe amplification process relative to amplification conditions resultingin completely extended strands.
 3. The method of claim 1, furthercomprising adding oligonucleotides which anneal with incompletelyextended amplified strands in the annealing step.
 4. The method of claim1, wherein a plurality of variants of a polynucleotide are substratesfor the polynucleotide extension process in step (a).
 5. A method ofproducing mutant polynucleotides, comprising:(a) conducting apolynucleotide extension process under conditions resulting inincomplete extension of synthesized polynucleotides wherein at least 20%of the amplified strands are incompletely extended; (b) subjectingamplification products to further amplification to produce a recombinantvariant of the polynucleotide.
 6. A method for producing mutantpolynucleotides, comprising:(a) conducting a polynucleotide extensionprocess under conditions resulting in incomplete extension ofsynthesized polynucleotides the process producing a plurality ofpolynucleotides in various stages of synthesis or amplification whereinat least 20% of the amplified strands are incompletely extended; (b)denaturing the plurality of polynucleotides; (c) incubating thedenatured plurality of polynucleotides with a polymerase underconditions which result in annealing of the single-strandedpolynucleotides at regions of identity between the single strandedpolynucleotides and formation of mutagenized double strandedpolynucleotide chains; (d) repeating (b) and (c); (e) expressing aplurality of mutant polypeptides from a plurality of mutagenizedpolynucleotide chains; (f) screening or selecting the plurality ofmutant polypeptides to identify at least one mutant polypeptide having auseful functional activity.
 7. The method of claim 6, further comprisingcloning the plurality of mutagenized polynucleotide chains into a vectorbefore the expressing step.
 8. A method of producing a variantpolynucleotide, comprising:(a) subjecting a plurality of variants of apolynucleotide to a template-directed amplification process underconditions resulting in incompletely extended amplification productswherein at least 20% of the amplified strands are incompletely extended;(b) denaturing the amplification products; (c) annealing the denaturedamplification products at regions of complementarity and incubating witha polymerase under conditions for template-directed amplification; (d)repeating (b) and (c) until the amplification products include aplurality of recombinant variants of the polynucleotide; and (e)screening or selecting a plurality of recombinant variants or expressionproducts thereof to identify a recombinant variant having a desiredfunctional property.
 9. The method of claim 8, wherein the conditionsresulting in incomplete extension are achieved by shortening theextension time of the amplification process relative to amplificationconditions resulting in completely extended amplified strands.
 10. Themethod of claim 8, further comprising adding oligonucleotides whichanneal with incompletely extended amplified strands in the annealingstep.
 11. A method of producing a recombinant polynucleotide,comprising:(a) choosing a plurality of existing polynucleotide variantsto be shuffled; (b) subjecting the plurality of polynucleotide variantsto a template-directed polynucleotide extension process under conditionsresulting in incomplete extension of the polynucleotide variants whereinat least 20% of the amplified strands are incompletely extended toproduce a plurality of single-stranded overlapping segments havingsequences from the plurality of polynucleotide variants; (c) conductinga multi-cyclic polynucleotide extension process on pairs of the singlestranded overlapping segments annealed to each other at region sooverlap under conditions whereby one segment serves as a template forextension of the other to generate a population of recombinant variantsof the polynucleotides; (d) screening or selecting the recombinantvariants or expression products thereof to identify a recombinantvariant having a desired functional property.
 12. A method of producinga shuffled polynucleotide, comprising:(a) choosing a plurality ofexisting polynucleotide variants to be shuffled; (b) generating partiallength copies of the plurality of variants by PCR amplification underconditions in which at least 20% of products are partial length productsthe partial length copies constituting a plurality of single-strandedoverlapping segments having sequences form the plurality ofpolynucleotide variants; (c) conducting a multi-cyclic polynucleotideextension process on pairs of the single stranded overlapping segmentsannealed to each other at region so overlap under conditions whereby onesegment serves as a template for extension of the other to generate apopulation of shuffled polynucleotides; (d) screening or selecting theshuffled variant polynucleotides or expression products thereof toidentify a shuffled variant polynucleotide or expression product thereofhaving a desired functional property.
 13. A method of evolving a variantpolynucleotide for acquisition of a desired property,comprisingconducting a polynucleotide amplification reaction on initialsubstrates comprising a plurality of variants of a polynucleotide,wherein at least one cycle of the amplification reaction is anincomplete amplification cycle performed under conditions which produceamplification products comprising incompletely extended variants of thepolynucleotide wherein at least 20% of the amplified strands areincompletely extended, which amplification products are denatured tocomponent strands, which are reannealed in different pairings to formrecombined amplification products, which form the substrates for asubsequent cycle of amplification until the amplification productsinclude evolved recombinant variants of the polynucleotide; selecting orscreening the evolved recombinant variants of the polynucleotide toidentify an evolved recombinant variant having a desired functionalproperty.
 14. The method of claim 13, wherein the conditions resultingin incomplete extension are achieved by shortening the extension time ofthe amplification process relative to amplification conditions resultingin complete extension of the variants of the polynucleotide.
 15. Themethod of claim 13, wherein the polynucleotide amplification reaction isconducted using taq polymerase and a second polymerase is added in theat least one cycle of incomplete amplification.
 16. A method of evolvinga variant polynucleotide for acquisition of a desired property,comprisingconducting a polynucleotide amplification reaction on initialsubstrates comprising a plurality of variants of a polynucleotide,wherein at least one cycle of the amplification reaction is anincomplete amplification cycle performed under conditions which produceamplification products comprising incompletely extended variants of thepolynucleotide, which amplification products are denatured to componentstrands, which are reannealed in different pairings to form recombinedamplification products, which form the substrates for a subsequent cycleof amplification until the amplification products include evolvedrecombinant variants of the polynucleotide; selecting or screening theevolved recombinant variants of the polynucleotide to identify arecombinant variant having a desired functional property wherein thepolynucleotide amplification reaction is conducted for 10-20 cycles ofincomplete amplification and for 10-20 cycles under conditionspermitting complete extension of the variants of the polynucleotide. 17.The method of claim 13, wherein the 10-20 cycles of incomplete extensionare performed using Klenow polymerase, and the 10-20 cycles underconditions permitting complete extension are performed using Taqpolymerase.
 18. The method of claim 13, wherein the at least one cycleof incomplete amplification is performed using a heat-labile polymerase.19. The method of claim 13, wherein the variants of the polynucleotidecomprises natural variants.
 20. The method of claim 13, wherein thevarients of the polynucleotide comprise allelic variants.
 21. The methodof claim 13, wherein the naturally occurring variants of apolynucleotide comprises species variants of a gene.
 22. The method ofclaim 13, wherein the desired property of the polynucleotide orexpression product is selected from the group consisting of capacity tobind a receptor, drug resistance, a therapeutic activity, suitabilityfor gene therapy, and suitability for use as a DNA-based vaccine. 23.The method of claim 13, further comprising choosing the initialsubstrates on which the polynucleotide amplification reaction isconducted.
 24. The method of claim 13 performed reiteratively, withscreened or selected polynucleotides from one cycle providing theinitial substrates for the next cycle.
 25. The method of claim 1,further comprising expressing the variant polynucleotide having theuseful functional activity to produce an expression product.
 26. Themethod of claim 25, wherein the variant polynucleotide is expressed in ahost cell.
 27. The method of claim 25, wherein the variantpolynucleotide encodes an enzyme.
 28. The method of claim 25, whereinthe variant polynucleotide encodes a viral coat protein.
 29. The methodof claim 1, wherein the screening or selecting step comprisesintroducingthe plurality of variant polynucleotides into a population of cells;expressing the variant polynucleotides in the population of cells; andselecting or screening the population of cell for the useful functionalactivity.
 30. The method of claim 5, further comprising expressing therecombinant variant of the polynucleotide.
 31. The method of claim 30,wherein the recombinant variant polynucleotide is expressed in a hostcell.
 32. The method of claim 30, wherein the recombinant variantpolynucleotide encodes an enzyme.
 33. The method of claim 30, whereinthe recombinant variant polynucleotide encodes a viral coat protein. 34.The method of claim 30, wherein the recombinant variant polynucleotideencodes an antibody chain.
 35. The method of claim 30, wherein therecombinant variant polynucleotide encodes a polymerase.
 36. The methodof claim 5, further comprising:introducing the recombinant variant intoa population of cells; expressing the recombinant variant in thepopulation of cells; and selecting or screening the population of cellsfor a useful functional activity.
 37. The method of claim 6, wherein theat least one mutant polypeptide is an enzyme.
 38. The method of claim 6,wherein the at least one mutant polypeptide is a viral coat protein. 39.The method of claim 6, wherein the at least one mutant polypeptide is anantibody chain.
 40. The method of claim 6, wherein the at least onemutant polypeptide is a polymerase.
 41. The method of claim 6, furthercomprising:introducing the mutagenized double stranded polynucleotidechains into a host cell before step (e).
 42. The method of claim 8 or11, further comprising expressing the recombinant variant having thedesired functional property.
 43. The method of claim 42, wherein therecombinant variant is expressed in a host cell.
 44. The method of claim42, wherein the recombinant variant encodes an enzyme.
 45. The method ofclaim 42, wherein the recombinant variant encodes a viral coat protein.46. The method of claim 42, wherein the recombinant variant encodes anantibody chain.
 47. The method of claim 42, wherein the recombinantvariant encodes a polymerase.
 48. The method of claim 8 or 11, whereinthe screening or selecting step comprisesintroducing the recombinantvariants into a population of cells; expressing the recombinant variantsin the population of cells; and selecting or screening the population ofcell for the useful functional activity.
 49. The method of claim 12,further comprising expressing the shuffled variant polynucleotide havingthe desired functional property.
 50. The method of claim 49, wherein theshuffled variant polynucleotide is expressed in a host cell.
 51. Themethod of claim 49, wherein the shuffled variant polynucleotide encodesan enzyme.
 52. The method of claim 49, wherein the shuffled variantpolynucleotide encodes a viral coat protein.
 53. The method of claim 49,wherein the shuffled variant polynucleotide encodes an antibody chain.54. The method of claim 49, wherein the shuffled variant polynucleotideencodes a polymerase.
 55. The method of claim 12, wherein the screeningor selecting step comprisesintroducing the shuffled variantpolynucleotides into a population of cells; expressing the shuffledvariant polynucleotides in the population of cells; and selecting orscreening the population of cells for the useful functional activity.56. The method of claim 13, further comprising expressing the evolvedrecombinant variant having the desired functional property.
 57. Themethod of claim 56, wherein the evolved recombinant variant is expressedin a host cell.
 58. The method of claim 56, wherein the evolvedrecombinant variant encodes an enzyme.
 59. The method of claim 56,wherein the evolved recombinant variant encodes a viral coat protein.60. The method of claim 56, wherein the evolved recombinant variantencodes an antibody chain.
 61. The method of claim 56, wherein theevolved recombinant variant encodes a polymerase.
 62. The method ofclaim 13, wherein the screening or selecting step comprisesintroducingthe evolved recombinant variants of the polynucleotide into a populationof cells; expressing the evolved recombinant variants of thepolynucleotide in the population of cells; and selecting or screeningthe population of cells for the desired functional activity.